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Research & Scholarship

Current Research and Scholarly Interests

Our primary interests are in understanding the molecular underpinnings of vascular disease as well as assessing disease risk. We use a wide range of biochemical, molecular and physiological techniques to make primary observations in cell systems as well as preclinical models. Furthermore, we continue to extend our findings to human subjects in order to confirm their clinical applicability. Current research projects include:

Mechanisms regulating atherosclerosis and abdominal aortic aneurysm disease: While single genes can have dramatic effects in cellular biology, it is becoming increasingly clear that vascular disease (and health) is regulated by the coordinated expression of gene cassettes or pathways. By monitoring expression patterns of the entire genome simultaneously, we can begin to identify networks of genes that work in concert to affect disease progression. Moreover, this approach can often implicate specific nexus genes that are at the center of larger networks and/or participate in multiple pathways. Additionally, we are investigating the role microRNAs, a newly discovered class of small RNA molecues, in orchestrating the activity of multiple genes during the course of disease.

Role of insulin resistance: Reduced activity of the endogenous hormone, insulin, is now recognized as a cardinal feature of type 2 diabetes and an independent risk factor for cardiovascular disease. We have investigated the effects of insulin resistance in several tissues and have recently focused our attention on adipose tissue biology and how it relates to CVD. Long known as a storage vehicle for excess calories, the fat cell is now recognized to be a factory of different products that can not only affect local activity, but can circulate in the blood as hormones and regulate many biological processes. For example, we have recently reported that the novel hormone, apelin, is produced by fat tissue and has important effects upon insulin resistance, obesity and diabetes, all of which have significant implications for cardiovascular disease.

Biomarkers for risk assessment: In addition to target identification, we are applying transcriptional profiling and pathway analysis for another important aspect of cardiovascular disease management--biomarker discovery. As the name connotes, a biomarker should be a good indication of the disease state and thereby allow for early detection as well as monitoring disease progression and, hopefully, efficacy of an applied therapy. Biomarkers can encompass a wide range of molecules including DNA variants, RNA, proteins, as well as lipids. They can even encompass modalities such as molecular imaging. We are engaged in not only identifying novel biomarkers for cardiovascular disease, but also in producing algorithms that combine multiple biomarkers to optimally assess risk.

Clinical Trials

The aim of the Antioxidant Study is to compare the efficacy of foods naturally rich in
antioxidants with that of antioxidants in a pill form on markers of inflammation and plasma
cholesterol in healthy adults at risk of cardiovascular disease.

Stanford is currently not accepting patients for this trial.For more information, please contact Antonella Dewell, (650) 736 - 8577.

An abdominal aortic aneurysm (AAA) is a weakened and enlarged area in the abdominal aorta,
which is a large blood vessel in the abdomen. If an AAA ruptures, it can be
life-threatening. Research has shown that sedentary individuals are at increased risk of
developing AAAs. This study will evaluate the effectiveness of an exercise program at
limiting the growth of small AAAs in older individuals.

Stanford is currently not accepting patients for this trial.For more information, please contact Ronald Dalman, (650) 723 - 2169.

Effects of Glutathione (an Antioxidant) and N-Acetylcysteine on InflammationNot Recruiting

The rationale for the potential role of antioxidants in the prevention of cardiovascular
diseases (CVD) remains strong despite the disappointing results of recent trials with a few
select antioxidant vitamins. Glutathione (GSH) is one of the body's most powerful
antioxidant agents but there is a surprising paucity of data on its use as an interventional
therapy.
Glutathione, when taken orally, is immediately broken down into its constituent amino acids,
of which cysteine is the only one to be essential. Available cysteine is the critical
determinant of intracellular GSH concentrations. N-acetyl cysteine (NAC) is an antioxidant
supplement that has been used to provide a source of cysteine to replete GSH levels. By
replenishing endogenous glutathione, it is possible that NAC would exert the same effect(s)
as exogenous GSH.
However, there is a new delivery system, liposomal GSH, which keeps glutathione intact. In
this study, the investigators propose to match the cysteine content of NAC and GSH and
compare the effects of these two supplements, at two different doses, on markers of
inflammation and oxidative stress.

Stanford is currently not accepting patients for this trial.For more information, please contact Antonella Dewell, (650) 736 - 8577.

Effects of Omega-3 Fatty Acids on Markers of InflammationNot Recruiting

The major purpose of this study is to examine the effect of two sources of dietary omega-3
fatty acids, each given at two doses, on potential health benefits related to cardiovascular
disease prevention. The two sources of dietary omega-3 fatty acids will be fish oil, and
flax seed oil.

Stanford is currently not accepting patients for this trial.For more information, please contact Antonella Dewell, (650) 736 - 8577.

To determine whether biomarkers assessed in blood samples can be used to detect individuals
at risk for developing blood clots or worsening of their underlying disease. The ultimate
goal of the study is to identify key biomarkers derived from blood that are most
characteristic and informative of individuals who will go on to develop a clotting
complication.

Stanford is currently not accepting patients for this trial.For more information, please contact Fizaa Ahmed, 650-725-6409.

Abstract

Abdominal aortic aneurysm (AAA) disease is a common, morbid, and highly lethal pathology. Extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of AAAs. Although surgery is highly effective in preventing death by rupture for larger AAAs, no guidance or preventive therapy is currently available for the >90% of patients whose aneurysms are below the surgical threshold. Predictive animal models of AAA as well as human pathological samples have revealed a complex circuit of AAA formation and progression. The proteolytic destruction of matrix components of the aorta by different proteases has been extensively studied over many years. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as "fine-tuners" of the translational output of target genes; they act by promoting mRNA degradation. Their therapeutic potential in limiting AAA development appears very intriguing. Further, current studies assessing genetic and heritable associations for AAA disease have provided great insight into its pathogenesis, potentially enabling us to better clinically manage affected patients.

Abstract

Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid-modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.

Abstract

MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

Abstract

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

Abstract

Epigenetic biomarkers of aging (the "epigenetic clock") have the potential to address puzzling findings surrounding mortality rates and incidence of cardio-metabolic disease such as: (1) women consistently exhibiting lower mortality than men despite having higher levels of morbidity; (2) racial/ethnic groups having different mortality rates even after adjusting for socioeconomic differences; (3) the black/white mortality cross-over effect in late adulthood; and (4) Hispanics in the United States having a longer life expectancy than Caucasians despite having a higher burden of traditional cardio-metabolic risk factors.We analyzed blood, saliva, and brain samples from seven different racial/ethnic groups. We assessed the intrinsic epigenetic age acceleration of blood (independent of blood cell counts) and the extrinsic epigenetic aging rates of blood (dependent on blood cell counts and tracks the age of the immune system). In blood, Hispanics and Tsimane Amerindians have lower intrinsic but higher extrinsic epigenetic aging rates than Caucasians. African-Americans have lower extrinsic epigenetic aging rates than Caucasians and Hispanics but no differences were found for the intrinsic measure. Men have higher epigenetic aging rates than women in blood, saliva, and brain tissue.Epigenetic aging rates are significantly associated with sex, race/ethnicity, and to a lesser extent with CHD risk factors, but not with incident CHD outcomes. These results may help elucidate lower than expected mortality rates observed in Hispanics, older African-Americans, and women.

Abstract

Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure.Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

Abstract

Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication.We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed.This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.

Abstract

Accelerated arterial stiffening is a major complication of diabetes mellitus with no specific therapy available to date.The present study investigates the role of the osteogenic transcription factor runt-related transcription factor 2 (Runx2) as a potential mediator and therapeutic target of aortic fibrosis and aortic stiffening in diabetes mellitus.Using a murine model of type 2 diabetes mellitus (db/db mice), we identify progressive structural aortic stiffening that precedes the onset of arterial hypertension. At the same time, Runx2 is aberrantly upregulated in the medial layer of db/db aortae, as well as in thoracic aortic samples from patients with type 2 diabetes mellitus. Vascular smooth muscle cell-specific overexpression of Runx2 in transgenic mice increases expression of its target genes, Col1a1 and Col1a2, leading to medial fibrosis and aortic stiffening. Interestingly, increased Runx2 expression per se is not sufficient to induce aortic calcification. Using in vivo and in vitro approaches, we further demonstrate that expression of Runx2 in diabetes mellitus is regulated via a redox-sensitive pathway that involves a direct interaction of NF-κB with the Runx2 promoter.In conclusion, this study highlights Runx2 as a previously unrecognized inducer of vascular fibrosis in the setting of diabetes mellitus, promoting arterial stiffness irrespective of calcification.

Abstract

Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

Abstract

Abdominal aortic aneurysms (AAA) are an important source of morbidity and mortality in the U.S. and worldwide. Treatment options are limited, with open surgery or endovascular repair remaining the only curative treatments. Classical cardiovascular medications have generally failed to prevent or significantly alter AAA formation or progression. Therefore, there is a tremendous need for better therapeutic approaches. With increasing knowledge of microRNA (miR) regulation in the context of cardiovascular disease, and with improving technical options permitting alteration of miR-expression levels in vitro and in vivo, we are offered a glimpse into the diagnostic and therapeutic possibilities of using miRs to treat vascular pathobiology. This review focuses on the role of miRs in aneurysmal disease of the abdominal aorta, summarizing recent publications regarding this topic, and outlining known effects of relevant miRs in AAA formation, including miR-21 and miR-29b. Despite there being only limited studies available, several other miRs also display clear potential for alteration of the disease process including miR-26a, the miR-17-92-cluster, miRs-221/222, miR-133 and miR-146a. While studies have shown that miRs can regulate the activity and interplay of vascular inflammatory cells, endothelial cells, smooth muscle cells and fibroblasts, all key elements leading to AAA formation, much work remains to be done.

Abstract

Treatment of decompensated heart failure often includes administration of levosimendan. Myeloperoxidase (MPO) is released during polymorphonuclear neutrophil (PMN) degranulation, and mediates dysregulation of vascular tone in heart failure. We evaluated the effects of levosimendan-treatment on MPO in patients with acute decompensation of chronic heart failure over a one week course. Plasma MPO levels were significantly decreased after levosimendan treatment (from 252.1 ± 31.1 pmol/l at baseline to 215.02 ± 27.96 pmol/l at 6 h, p < 0.05). Ex vivo incubation of whole blood with levosimendan decreased MPO release after PMN-stimulation (8.2 ± 1.4-fold increase at baseline vs. 6.0 ± 1.1-fold increase with levosimendan). MPO levels also significantly correlated with diastolic blood pressure over the time course. In a multivariate linear model, the main contributor to systolic, diastolic and mean blood pressure was level of PMN elastase. MPO contributed only in heparin-treated patients, suggesting a more significant role for endothelial-bound MPO than for circulating MPO or elastase with respect to blood pressure regulation. We here provide the first evidence that levosimendan treatment inhibits MPO release by PMNs in decompensated heart failure patients. This mechanism may regulate endothelial function and vascular tone in heart failure patients.

Abstract

Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme.

Abstract

Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.

Abstract

Significance: Arterial blood vessels functionally and structurally adapt to altering hemodynamic forces in order to accommodate changing needs and to provide stress homeostasis. This ability is achieved at the cellular level by converting mechanical stimulation into biochemical signals (i.e., mechanotransduction). Whereas physiological mechanical stress helps to maintain vascular structure and function, pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes which drive disease. Recent advances: Reactive oxygen species (ROS) may represent an intriguing class of mechanically- regulated second messengers. Chronically enhanced ROS-generation may be induced by adverse mechanical stresses, and is associated with a multitude of vascular diseases. Although a causal relationship has clearly been demonstrated in large numbers of animal studies, an effective ROS-modulating therapy still remains to be established by clinical studies. Critical issues and Future directions: This review article focuses on the role of various mechanical forces (in the form of laminar shear stress, oscillatory shear stress or cyclic stretch) as modulators of ROS- driven signaling, and their subsequent effects on vascular biology and homeostasis, as well as on specific diseases such as arteriosclerosis, hypertension and abdominal aortic aneurysms. Specifically, it highlights the significance of the various NADPH oxidase (NOX) isoforms as critical ROS generators in the vasculature. Directed targeting of defined components in the complex network of ROS (mechano)signaling may represent a key for successful translation of experimental findings into clinical practice.

Abstract

Tremendous efforts have been initiated to elucidate the molecular and pathophysiological characteristics of abdominal aortic aneurysm (AAA) disease, which is a significant contributor to morbidity and mortality in the Western world. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as important transcriptional and posttranscriptional inhibitors of gene expression thought to simultaneously "fine tune" the translational output of multiple target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. Several research groups were able to identify the miR-29 family, and miR-29b in particular, as crucial regulators of-not only vascular fibrosis-but also cardiac-, kidney-, liver-, and skin-fibrosis. The current review briefly points out data indicating a causal role for miR-29 in various diseases, while focusing on its potential benefit during AAA initiation and propagation.

Abstract

Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

Abstract

The contribution of abdominal aortic aneurysm (AAA) disease to human morbidity and mortality has increased in the aging, industrialized world. In response, extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of the diseased aorta. This work aims to develop novel diagnostic and therapeutic strategies to limit AAA expansion and, ultimately, rupture. Contributions from multiple research groups have uncovered a complex transcriptional and post-transcriptional regulatory milieu, which is believed to be essential for maintaining aortic vascular homeostasis. Recently, novel small noncoding RNAs, called microRNAs, have been identified as important transcriptional and post-transcriptional inhibitors of gene expression. MicroRNAs are thought to "fine tune" the translational output of their target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. With the discovery that microRNAs act as powerful regulators in the context of a wide variety of diseases, it is only logical that microRNAs be thoroughly explored as potential therapeutic entities. This current review summarizes interesting findings regarding the intriguing roles and benefits of microRNA expression modulation during AAA initiation and propagation. These studies utilize disease-relevant murine models, as well as human tissue from patients undergoing surgical aortic aneurysm repair. Furthermore, we critically examine future therapeutic strategies with regard to their clinical and translational feasibility.

Abstract

Two direct measurements of peripheral insulin sensitivity are the M value derived from the euglycemic, hyperinsulinemic clamp (EC) and the steady-state plasma glucose (SSPG) concentration derived from the insulin suppression test (IST). Prior work suggests that these measures are highly correlated, but the agreement between them is unknown. To determine the agreement between SSPG and M and to develop transformation equations to convert SSPG to M and vice versa, we directly compared these two measurements in the same individuals.A total of 15 nondiabetic subjects (9 women and 6 men) underwent both an EC and a modified version of the IST within a median interval of 5days. We performed standard correlation metrics of the two measures and developed transformation regression equations for the two measures.The mean±SD age of the subjects was 57±7years and body mass index, 27.7±3.9kg/m(2). The median (interquartile range) SSPG concentration was 6.7 (5.1, 9.8) mmol/L and M value, 49.6 (28.9, 64.2) μmol/min/kg-LBM. There was a highly significant correlation between SSPG and M (r=-0.87, P <0.001). The relationship was best fit by regression models with exponential/logarithmic functions (R(2)=0.85). Bland-Altman plots demonstrated an excellent agreement between these measures of insulin action.The SSPG and M are highly related measures of insulin sensitivity and the results provide the means to directly compare the two measurements.

Abstract

Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear.Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model.These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

Abstract

In our previous transcriptional profiling of a murine model, we have identified a remarkably small number of specific pathways with altered expression in lymphedema. In this investigation, we utilized microarray-based transcriptomics of human skin for an unbiased a priori prospective candidate identification, with subsequent validation of these candidates through direct serum assay. The resulting multi-analyte biomarker panel sensitively should sensitively discriminate human lymphedema subjects from normal individuals.We enrolled 63 lymphedema subjects and 27 normals in our attempt to discover protein analytes that can distinguish diseased individuals from controls. To minimize technical and biologically irrelevant variation, we first identified potential candidates by performing transcriptional microarray analysis on paired diseased and normal skin specimens sampled from the same individuals. We focused our attention on genes with corresponding protein products that are secreted and took these candidates forward to a protein multiplex assay applied to diseased and normal subjects. We developed a logistic regression-based model on an eventual group of six proteins and validated our system on a separate cohort of study subjects. The area under the receiver operating characteristic curve was calculated to be 0.87 (95% CI : 0.75 to 0.97).We have developed an accurate bioassay utilizing proteins representing four central pathogenetic modalities of the disease: lymphangiogenesis, inflammation, fibrosis, and lipid metabolism, suggesting that these proteins are directly related to the pathogenesis of the tissue pathology in lymphatic vascular insufficiency. Further studies are warranted to determine whether this newly-identified biomarker panel will possess utility as an instrument for in vitro diagnosis of early and latent disease; the ultimate applicability to risk stratification, quantitation of disease burden, and response to therapy can easily be envisioned.

Abstract

Previous clinical studies in pulmonary arterial hypertension (PAH) have concentrated predominantly on distal pulmonary vascular resistance, its contribution to the disease process, and response to therapy. However, it is well known that biomechanical factors such as shear stress have an impact on endothelial health and dysfunction in other parts of the vasculature. This study tested the hypothesis that wall shear stress is reduced in the proximal pulmonary arteries of PAH patients with the belief that reduced shear stress may contribute to pulmonary endothelial cell dysfunction and as a result, PAH progression. A combined MRI and computational fluid dynamics (CFD) approach was used to construct subject-specific pulmonary artery models and quantify flow features and wall shear stress (WSS) in five PAH patients with moderate-to-severe disease and five age- and sex-matched controls. Three-dimensional model reconstruction showed PAH patients have significantly larger main, right, and left pulmonary artery diameters (3.5 ± 0.4 vs. 2.7 ± 0.1 cm, P = 0.01; 2.5 ± 0.4 vs. 1.9 ± 0.2 cm, P = 0.04; and 2.6 ± 0.4 vs. 2.0 ± 0.2 cm, P = 0.01, respectively), and lower cardiac output (3.7 ± 1.2 vs. 5.8 ± 0.6 L/min, P = 0.02.). CFD showed significantly lower time-averaged central pulmonary artery WSS in PAH patients compared to controls (4.3 ± 2.8 vs. 20.5 ± 4.0 dynes/cm(2), P = 0.0004). Distal WSS was not significantly different. A novel method of measuring WSS was utilized to demonstrate for the first time that WSS is altered in some patients with PAH. Using computational modeling in patient-specific models, WSS was found to be significantly lower in the proximal pulmonary arteries of PAH patients compared to controls. Reduced WSS in proximal pulmonary arteries may play a role in the pathogenesis and progression of PAH. This data may serve as a basis for future in vitro studies of, for example, effects of WSS on gene expression.

Abstract

Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-?), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.

Abstract

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches (1,2). The commonly used animal models include mice, rats, rabbits, and pigs (3-5). However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models (6,7). In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents. This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Abstract

Chronic inflammation is considered to play a role in the development of cardiovascular disease. Various (n-3) fatty acids (FA) have been reported to have antiinflammatory effects, but there is a lack of consensus in this area, particularly in regard to optimal source(s) and dose(s). This study aimed to determine the effects of high and low doses of (n-3) FA from plant and marine sources on plasma inflammatory marker concentrations. One-hundred adults with metabolic syndrome were randomly assigned to a low or high dose of plant- (2.2 or 6.6 g/d ?-linolenic acid) or marine- (1.2 or 3.6 g/d EPA and DHA) derived (n-3) FA or placebo for 8 wk, using a parallel arm design (n = 20/arm). Fasting blood samples collected at 0, 4, and 8 wk were analyzed for concentrations of monocyte chemotactic protein-1 (MCP-1), IL-6, and soluble intercellular adhesion molecule-1 (sICAM-1) and for cardiovascular risk factors. Baseline concentrations across all 5 groups combined were (mean ± SD) 103 ± 32 ng/L for MCP-1, 1.06 ± 0.56 ng/L for IL-6, and 0.197 ± 0.041 ng/L for sICAM-1. There were no significant differences in 8-wk changes in plasma inflammatory marker concentrations among the 5 groups. Plasma TG and blood pressure decreased significantly more and the LDL cholesterol concentration increased more in the high-dose fish oil group compared to the 8-wk changes in some of the other 4 groups (P ? 0.04). In conclusion, no beneficial effects were detected for any of the 3 inflammatory markers investigated in response to (n-3) FA in adults with metabolic syndrome regardless of dose or source.

Abstract

Although human embryonic stem cells (hESC) have enormous potential for cell replacement therapy of heart failure, immune rejection of hESC derivatives inevitably would occur after transplantation. We therefore aimed to generate a hypoantigeneic hESC line with improved survival characteristics.Using various in vivo, nonischemic, hindlimb xenotransplant models (immunocompetent and defined immunodefective mouse strains) as well as human in vitro T-cell and natural killer (NK)-cell assays, we revealed a central role for T cells in mediating hESC rejection. The NK-cell susceptibility of hESC in vivo was found to be low, and the NK response to hESC challenge in vitro was negligible. To reduce the antigenicity of hESC, we successfully generated human leukocyte antigen (HLA) I knockdown cells (hESC(siRNA+IB)) using both HLA I RNA interference (siRNA) and intrabody (IB) technology. HLA I expression was ?99% reduced after 7 days and remained low for weeks. Cellular immune recognition of these hESC(siRNA+IB) was strongly reduced in both xenogeneic and allogeneic settings. Immune rejection was profoundly mitigated after hESC(siRNA+IB) transplantation into immunocompetent mice, and even long-term graft survival was achieved in one third of the animals without any immunosuppression. The survival benefit of hESC(siRNA+IB) was further confirmed under ischemic conditions in a left anterior descending coronary artery ligation model.HLA I knockdown hESC(siRNA+IB) provoke T-cell ignorance and experience largely mitigated xenogeneic rejection. By generating hypoantigeneic hESC lines, the generation of acceptable hESC derivatives may become a practical concept and push cell replacement strategies forward.

Abstract

Apelin is a newly discovered peptide hormone that has recently been linked to insulin resistance and obesity. Data collected from both the clinical and basic research settings show that apelin: (i) is correlated with the states of insulin resistance and obesity; (ii) stimulates glucose utilization; (iii) decreases insulin secretion; and (iv) negatively regulates catecholamine-mediated lipolysis. These and other lines of evidence demonstrate that apelin may be a potentially viable candidate in the search for treatments for Type 2 diabetes and the insulin resistance (metabolic syndrome). The present review summarizes the literature on the regulation by apelin of glucose and lipid metabolism and the signaling pathways involved.

Abstract

Human embryonic stem cells (hESCs) can serve as a universal cell source for emerging cell or tissue replacement strategies, but immune rejection of hESC derivatives remains an unsolved problem. Here, we sought to describe the mechanisms of rejection for naïve hESCs and upon HLA class I (HLA I) knockdown (hESC(KD)). hESCs were HLA I-positive but negative for HLA II and co-stimulatory molecules. Transplantation of naïve hESC into immunocompetent Balb/c mice induced substantial T helper cell 1 and 2 (Th1 and Th2) responses with rapid cell death, but hESCs survived in immunodeficient SCID-beige recipients. Histology revealed mainly macrophages and T cells, but only scattered natural killer (NK) cells. A surge of hESC-specific antibodies against hESC class I, but not class II antigens, was observed. Using HLA I RNA interference and intrabody technology, HLA I surface expression of hESC(KD) was 88%-99% reduced. T cell activation after hESC(KD) transplantation into Balb/c was significantly diminished, antibody production was substantially alleviated, the levels of graft-infiltrating immune cells were reduced and the survival of hESC(KD) was prolonged. Because of their very low expression of stimulatory NK ligands, NK-susceptibility of naïve hESCs and hESC(KD) was negligible. Thus, HLA I recognition by T cells seems to be the primary mechanism of hESC recognition, and T cells, macrophages and hESC-specific antibodies participate in hESC killing.

Abstract

The lipid-lowering agent pravastatin and the antidepressant paroxetine are among the most widely prescribed drugs in the world. Unexpected interactions between them could have important public health implications. We mined the US Food and Drug Administration's (FDA's) Adverse Event Reporting System (AERS) for side-effect profiles involving glucose homeostasis and found a surprisingly strong signal for comedication with pravastatin and paroxetine. We retrospectively evaluated changes in blood glucose in 104 patients with diabetes and 135 without diabetes who had received comedication with these two drugs, using data in electronic medical record (EMR) systems of three geographically distinct sites. We assessed the mean random blood glucose levels before and after treatment with the drugs. We found that pravastatin and paroxetine, when administered together, had a synergistic effect on blood glucose. The average increase was 19 mg/dl (1.0 mmol/l) overall, and in those with diabetes it was 48 mg/dl (2.7 mmol/l). In contrast, neither drug administered singly was associated with such changes in glucose levels. An increase in glucose levels is not a general effect of combined therapy with selective serotonin reuptake inhibitors (SSRIs) and statins.

Abstract

Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-?-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P?0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-? pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.

Abstract

To quantitatively compare aortic curvature and motion with resulting aneurysm location, direction of expansion, and pathophysiological features in experimental abdominal aortic aneurysms (AAAs).MRI was performed at 4.7 T with the following parameters: (1) 3D acquisition for vessel geometry and (2) 2D cardiac-gated acquisition to quantify luminal motion. Male 24-week-old mice were imaged before and after AAA formation induced by angiotensin II (AngII)-filled osmotic pump implantation or infusion of elastase. AngII-induced AAAs formed near the location of maximum abdominal aortic curvature, and the leftward direction of expansion was correlated with the direction of suprarenal aortic motion. Elastase-induced AAAs formed in a region of low vessel curvature and had no repeatable direction of expansion. AngII significantly increased mean blood pressure (22.7 mm Hg, P<0.05), whereas both models showed a significant 2-fold decrease in aortic cyclic strain (P<0.05). Differences in patterns of elastin degradation and localization of fluorescent signal from protease-activated probes were also observed.The direction of AngII aneurysm expansion correlated with the direction of motion, medial elastin dissection, and adventitial remodeling. Anterior infrarenal aortic motion correlated with medial elastin degradation in elastase-induced aneurysms. Results from both models suggest a relationship between aneurysm pathological features and aortic geometry and motion.

Abstract

Atherosclerosis is a leading cause of death worldwide. Macrophages are key components of vascular inflammation, which contributes to the development and complications of atherosclerosis. Ferritin, an iron storage and transport protein, has been found to accumulate in macrophages in human atherosclerotic plaques. We hypothesized that ferritin could serve as an intrinsic nano-platform to target delivery of imaging agents to vascular macrophages to detect high-risk atherosclerotic plaques. Here we show that engineered human ferritin protein cages, either conjugated to the fluorescent Cy5.5 molecule or encapsulating a magnetite nanoparticle, are taken up in vivo by macrophages in murine atherosclerotic carotid arteries and can be imaged by fluorescence and magnetic resonance imaging. These results indicate that human ferritin can serve as a nanoparticle platform to image vascular inflammation in vivo.

Abstract

The release of free fatty acids (FFAs) from adipocytes (i.e. lipolysis) is increased in obesity and is a contributory factor to the development of insulin resistance. A recently identified adipokine, apelin, is up-regulated in states of obesity. Although apelin is secreted by adipocytes, its functions in them remain largely unknown. To determine whether apelin affects lipolysis, FFA, glycerol, and leptin levels, as well as abdominal adiposity, were measured at baseline and after reintroduction of exogenous apelin in apelin-null mice. To examine apelin's effects in vitro, isoproterenol-induced FFA/glycerol release, and hormone-sensitive lipase (HSL) and acetyl CoA carboxylase phosphorylation were investigated in 3T3-L1 cells and isolated wild-type adipocytes. Serum FFA, glycerol, and leptin concentrations, as well as abdominal adiposity, were significantly increased in apelin-null vs. wild-type mice; these changes were ameliorated in response to exogenous apelin. Apelin also reduced isoproterenol-induced FFA release in adipocytes isolated from wild-type but not APJ-null mice. In 3T3-L1 cells and isolated adipocytes, apelin attenuated isoproterenol-induced FFA/glycerol release. Apelin's inhibition was reversed by pertussis toxin, the G(q) inhibitor glycoprotein antagonist 2A, and the AMP-activated protein kinase inhibitors compound C and dorsomorphin. Apelin increased HSL phosphorylation at Ser-565 and also abrogated isoproterenol-induced HSL phosphorylation at Ser-563. Notably, apelin increased acetyl CoA carboxylase phosphorylation, suggesting AMPK activation. In conclusion, apelin negatively regulates lipolysis. Its actions may be mediated by pathways involving G(q), G(i), and AMP-activated protein kinase.

Abstract

The biomechanical forces associated with blood flow have been shown to play a role in pulmonary vascular cell health and disease. Therefore, the quantification of human pulmonary artery hemodynamic conditions under resting and exercise states can be useful in investigating the physiology of disease development and treatment outcomes. In this study, a combined magnetic resonance imaging and computational fluid dynamics approach was used to quantify pulsatile flow fields, wall shear stress (WSS), oscillations in WSS (OSI), and energy efficiency in six subject-specific models of the human pulmonary vasculature with high spatial and temporal resolution. Averaging over all subjects, WSS was found to increase from 19.8±4.0 to 51.8±6.7 dynes/cm2, and OSI was found to decrease from 0.094±0.016 to 0.081±0.015 in the proximal pulmonary arteries between rest and exercise conditions (p<0.05). These findings demonstrate the localized, biomechanical effects of exercise. Furthermore, an average decrease of 10% in energy efficiency was noted between rest and exercise. These data indicate the amount of energy dissipation that typically occurs with exercise and may be useful in future surgical planning applications.

Abstract

The aim of this study was to definitively assess the validity of noninvasive high-frequency ultrasound (US) measurements of aortic luminal diameter (ALD) in a murine model of elastase-induced abdominal aortic aneurysm in comparison with in situ video microscopy (VM).C57BL/6 mice underwent transient perfusion of the aorta with either elastase (n = 20: Elastase group) or saline (n = 10: Sham). Unoperated mice (n = 10) were also studied.ALD measurements by US had excellent linear correlation and absolute agreement with that by VM in both Control (unoperated or sham-operated mice) and elastase groups (r = 0.96, intraclass correlation coefficient (ICC) = 0.88 and r = 0.93, ICC = 0.92, resp.). Bland-Altman analysis of US compared with VM measurements in both groups indicated good agreement, however US measurements were slightly but significantly higher than VM measurements in the control group (mean bias 0.039?mm, P < .05). Linear regression analysis revealed excellent correlation between US and VM measurements in both groups. (R² = 0.91 in Control group, R² = 0.85 in elastase group.) The reliability of US measurements was also confirmed by ex vivo histological measurements.High-frequency US provides reliable ALD measurements in developing murine abdominal aortic aneurysms.

Abstract

To develop methods to quantify cyclic strain, motion, and curvature of the murine abdominal aorta in vivo.C57BL/6J and apoE(-/-) mice underwent three-dimensional (3D) time-of-flight MR angiography to position cardiac-gated 2D slices at four locations along the abdominal aorta where circumferential cyclic strain and lumen centroid motion were calculated. From the 3D data, a centerline through the aorta was created to quantify geometric curvature at 0.1-mm intervals. Medial elastin content was quantified with histology postmortem. The location and shape of abdominal aortic aneurysms (AAAs), created from angiotensin II infusion, were evaluated qualitatively.Strain waveforms were similar at all locations and between groups. Centroid motion was significantly larger and more leftward above the renal vessels than below (P < 0.05). Maximum geometric curvature occurred slightly proximal to the right renal artery. Elastin content was similar around the circumference of the vessel. AAAs developed in the same location as the maximum curvature and grew in the same direction as vessel curvature and motion.The methods presented provide temporally and spatially resolved data quantifying murine aortic motion and curvature in vivo. This noninvasive methodology will allow serial quantification of how these parameters influence the location and direction of AAA growth.

Abstract

We recently reported that a preponderance of small adipose cells, decreased expression of cell differentiation markers, and enhanced inflammatory activity in human subcutaneous whole adipose tissue were associated with insulin resistance. To test the hypothesis that small adipocytes exhibited these differential properties, we characterised small adipocytes from epididymal adipose tissue of Zucker Obese (ZO) and Lean (ZL) rats. Rat epididymal fat pads were removed and adipocytes isolated by collagenase digestion. Small adipocytes were separated by sequential filtration through nylon meshes. Adipocytes were fixed in osmium tetroxide for cell size distribution analysis via Beckman Coulter Multisizer. Quantitative real-time PCR for cell differentiation and inflammatory genes was performed. Small adipocytes represented a markedly greater percentage of the total adipocyte population in ZO than ZL rats (58±4% vs. 12±3%, p<0.001). In ZO rats, small as compared with total adipocytes had 4-fold decreased adiponectin, and 4-fold increased visfatin and IL-6 levels. Comparison of small adipocytes in ZO versus ZL rats revealed 3-fold decreased adiponectin and PPAR? levels, and 2.5-fold increased IL-6. In conclusion, ZO rat adipose tissue harbours a large proportion of small adipocytes that manifest impaired cell differentiation and pro-inflammatory activity, two mechanisms by which small adipocytes may contribute to insulin resistance.

Abstract

Rodent and in vitro studies suggest that thiazolidinediones promote adipogenesis but there are few studies in humans to corroborate these findings. The purpose of this study was to determine whether pioglitazone stimulates adipogenesis in vivo and whether this process relates to improved insulin sensitivity. To test this hypothesis, 12 overweight/obese nondiabetic, insulin-resistant individuals underwent biopsy of abdominal subcutaneous adipose tissue at baseline and after 12 weeks of pioglitazone treatment. Cell size distribution was determined via the Multisizer technique. Insulin sensitivity was quantified at baseline and postpioglitazone by the modified insulin suppression test. Regional fat depots were quantified by computed tomography (CT). Insulin resistance (steady-state plasma insulin and glucose (SSPG)) decreased following pioglitazone (P < 0.001). There was an increase in the ratio of small-to-large cells (1.16 +/- 0.44 vs. 1.52 +/- 0.66, P = 0.03), as well as a 25% increase in the absolute number of small cells (P = 0.03). The distribution of large cell diameters widened (P = 0.009), but diameter did not increase in the case of small cells. The increase in proportion of small cells was associated with the degree to which insulin resistance improved (r = -0.72, P = 0.012). Visceral abdominal fat decreased (P = 0.04), and subcutaneous abdominal (P = 0.03) and femoral fat (P = 0.004) increased significantly. Changes in fat volume were not associated with SSPG change. These findings demonstrate a clear effect of pioglitazone on human subcutaneous adipose cells, suggestive of adipogenesis in abdominal subcutaneous adipose tissue, as well as redistribution of fat from visceral to subcutaneous depots, highlighting a potential mechanism of action for thiazolidinediones. These findings support the hypothesis that defects in subcutaneous fat storage may underlie obesity-associated insulin resistance.

Abstract

Although they have become a widely used experimental technique for identifying differentially expressed (DE) genes, DNA microarrays are notorious for generating noisy data. A common strategy for mitigating the effects of noise is to perform many experimental replicates. This approach is often costly and sometimes impossible given limited resources; thus, analytical methods are needed which increase accuracy at no additional cost. One inexpensive source of microarray replicates comes from prior work: to date, data from hundreds of thousands of microarray experiments are in the public domain. Although these data assay a wide range of conditions, they cannot be used directly to inform any particular experiment and are thus ignored by most DE gene methods. We present the SVD Augmented Gene expression Analysis Tool (SAGAT), a mathematically principled, data-driven approach for identifying DE genes. SAGAT increases the power of a microarray experiment by using observed coexpression relationships from publicly available microarray datasets to reduce uncertainty in individual genes' expression measurements. We tested the method on three well-replicated human microarray datasets and demonstrate that use of SAGAT increased effective sample sizes by as many as 2.72 arrays. We applied SAGAT to unpublished data from a microarray study investigating transcriptional responses to insulin resistance, resulting in a 50% increase in the number of significant genes detected. We evaluated 11 (58%) of these genes experimentally using qPCR, confirming the directions of expression change for all 11 and statistical significance for three. Use of SAGAT revealed coherent biological changes in three pathways: inflammation, differentiation, and fatty acid synthesis, furthering our molecular understanding of a type 2 diabetes risk factor. We envision SAGAT as a means to maximize the potential for biological discovery from subtle transcriptional responses, and we provide it as a freely available software package that is immediately applicable to any human microarray study.

Abstract

Inflammation is associated with increased body mass and purportedly with increased size of adipose cells. We sought to determine whether increased size of adipose cells is associated with localised inflammation in weight-stable, moderately obese humans.We recruited 49 healthy, moderately obese individuals for quantification of insulin resistance (modified insulin suppression test) and subcutaneous abdominal adipose tissue biopsy. Cell size distribution was analysed with a multisizer device and inflammatory gene expression with real-time PCR. Correlations between inflammatory gene expression and cell size variables, with adjustment for sex and insulin resistance, were calculated.Adipose cells were bimodally distributed, with 47% in a 'large' cell population and the remainder in a 'small' cell population. The median diameter of the large adipose cells was not associated with expression of inflammatory genes. Rather, the fraction of small adipose cells was consistently associated with inflammatory gene expression, independently of sex, insulin resistance and BMI. This association was more pronounced in insulin-resistant than insulin-sensitive individuals. Insulin resistance also independently predicted expression of inflammatory genes.This study demonstrates that among moderately obese, weight-stable individuals an increased proportion of small adipose cells is associated with inflammation in subcutaneous adipose tissue, whereas size of mature adipose cells is not. The observed association between small adipose cells and inflammation may reflect impaired adipogenesis and/or terminal differentiation. However, it is unclear whether this is a cause or consequence of inflammation. This question and whether small vs large adipose cells contribute differently to inflammation in adipose tissue are topics for future research. Trial registration: ClinicalTrials.gov NCT00285844.

Abstract

Thrombosis, the localized clotting of blood, occurs in both the arterial and venous circulation, and has a major impact on health outcomes. The primary etiology of myocardial infarctions, and approximately 80% of strokes, is acute arterial thrombosis. In combination this represents the most common cause of death in the Western world, while the third leading cause of cardiovascular-associated death is venous thromboembolism. An understanding of the pathogenic changes in the vessel wall and the blood that result in thrombosis is crucial for developing safer and more effective antithrombotic drugs. Dabigatran etexilate belongs to a new class of direct thrombin inhibitors. Following oral administration, dabigatran reaches peak plasma concentrations within 2 hours, shows linear pharmacokinetics, and a limited (but important) amount of direct drug interactions. Given once daily at 150 mg or 220 mg, it has proven to be competitive with enoxaparin in the prevention of venous thromboembolism after major orthopedic surgery, with a comparable safety profile. For stroke prevention in patients suffering from atrial fibrillation, dabigatran administered at a dose of 110 mg twice daily was associated with rates of stroke and systemic embolism that were similar to those associated with warfarin, as well as lower rates of hemorrhage. Dabigatran given at a dose of 150 mg twice daily, as compared with warfarin, was associated with lower rates of stroke and systemic embolism but similar rates of major hemorrhage. Oral bioavailability of dabigatran, together with a rapid onset and offset of action and predictable anticoagulation response, makes this newly available antithrombotic drug an attractive alternative to traditional anticoagulant therapies for numerous thrombosis-related indications.

Abstract

The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.

Abstract

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. The prevalence of AF increases sharply in old age (prevalence approximately 10% among persons 80 years of age and older). The expected risk for ischemic stroke is increased five-fold by the presence of AF, primarily as a result of cardiogenic embolism. Multiple large-scale, randomized trials have been completed or are still underway to find optimal, efficacious, and relatively safe ways to reduce the risk of ischemic stroke and other systemic thromboembolic events related to AF. Antithrombotic strategies are accompanied by serious bleeding complications that threaten patients in need of medical stroke prevention. Treatment regimens for preventing thromboembolism in AF patients range from vitamin K antagonists such as warfarin or coumadins, antiplatelet drugs like aspirin or clopidogrel, to newly developed orally available antithrombotics like the direct thrombin inhibitor dabigatran, or the Factor Xa-inhibitor rivaroxaban. The available anticoagulant and antiplatelet drugs have different advantages and disadvantages. This review attempts to delineate the specific role of clopidogrel in patients with AF and at risk of stroke, taking into consideration new and ongoing trials in this important field of medical practice.

Abstract

Endothelium-derived nitric oxide plays a crucial role in the regulation of vascular tone and the development of cardiovascular disease. The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) has emerged as a novel cardiovascular risk factor. ADMA appears to be an independent predictor for cardiovascular and overall mortality. However, the majority of studies investigating the clinical role of ADMA were performed in European study populations with few individuals of other ethnicities.We performed a cross-sectional study of 980 healthy, older (age 60-72 years) individuals of different ethnicities living in the San Francisco Bay area and analyzed ADMA plasma concentrations and their relationship to other cardiovascular risk factors. Plasma ADMA concentrations were measured using a recently developed, highly sensitive ELISA.In our entire sample, we were able to define a reference interval for ADMA plasma concentrations of 0.47 (90% CI 0.46-0.48) mumol/L to 0.85 (0.84-0.89) mumol/L. The mean ADMA concentration was 0.63 (SD 0.11) mumol/L (median 0.61 mumol/L). Mean ADMA concentrations were significantly lower in African Americans (0.60 mumol/L; P < 0.01) and mixed non-Hispanics (0.60 mumol/L; P < 0.05) compared with whites (0.63 mumol/L). ADMA was positively correlated with cystatin-C in both men (rho = 0.29) and women (rho = 0.37), and median plasma ADMA concentrations increased across cystatin-C quintiles.ADMA varies nearly 2-fold across a healthy sample of older men and women, correlates with age, body mass index, and renal function, and is different across ethnic groups. Additional studies in a wider age range and including larger ethnic subgroups would be useful.

Abstract

We recently identified differences in abdominal subcutaneous adipose tissue (SAT) from insulin-resistant (IR) as compared to obesity-matched insulin sensitive individuals, including accumulation of small adipose cells, decreased expression of cell differentiation markers, and increased inflammatory activity. This study was initiated to see if these changes in SAT of IR individuals were present in omental visceral adipose tissue (VAT); in this instance, individuals were chosen to be IR but varied in degree of adiposity. We compared cell size distribution and genetic markers in SAT and VAT of IR individuals undergoing bariatric surgery.Eleven obese/morbidly obese women were IR by the insulin suppression test. Adipose tissue surgical samples were fixed in osmium tetroxide for cell size analysis via Beckman Coulter Multisizer. Quantitative real-time polymerase chain reaction for genes related to adipocyte differentiation and inflammation was performed.While proportion of small cells and expression of adipocyte differentiation genes did not differ between depots, inflammatory genes were upregulated in VAT. Diameter of SAT large cells correlated highly with increasing proportion of small cells in both SAT and VAT (r = 0.85, p = 0.001; r = 0.72, p = 0.01, respectively). No associations were observed between VAT large cells and cell size variables in either depot. The effect of body mass index (BMI) on any variables in both depots was negligible.The major differential property of VAT of IR women is increased inflammatory activity, independent of BMI. The association of SAT adipocyte hypertrophy with hyperplasia in both depots suggests a primary role SAT may have in regulating regional fat storage.

Abstract

We previously reported an attenuation of both exercise hyperemia and measures of aerobic capacity in hypercholesterolemic mice. In this study, we expanded upon the previous findings by examining the temporal and quantitative relationship of hypercholesterolemia to aerobic and anaerobic capacity and by exploring several potential mechanisms of dysfunction. Eight-week-old wild type (n = 123) and apoE knockout (n = 79) C57BL/6J mice were divided into groups with distinct cholesterol levels by feeding with regular or high-fat diets. At various ages, the mice underwent treadmill ergospirometry. To explore mechanisms, aortic ring vasodilator function and nitrate (NO(x)) activity, urinary excretion of NO(x), running muscle microvascular density and citrate synthase activity, as well as myocardial mass and histologic evidence of ischemia were measured. At 8 weeks of age, all mice had similar measures of exercise capacity. All indices of aerobic exercise capacity progressively declined at 12 and 20 weeks of age in the hypercholesterolemic mice as cholesterol levels increased while indices of anaerobic capacity remained unaffected. Across the four cholesterol groups, the degree of aerobic dysfunction was related to serum cholesterol levels; a relationship that was maintained after correcting for confounding factors. Associated with the deterioration in exercise capacity was a decline in measures of nitric oxide-mediated vascular function while there was no evidence of aberrations in functional or oxidative capacities or in other components of transport capacity. In conclusion, aerobic exercise dysfunction is observed in murine models of genetic and diet-induced hypercholesterolemia and is associated with a reduction in vascular nitric oxide production.

Abstract

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.(1) The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.(2) The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Abstract

We have previously described differences in adipose cell size distribution and expression of genes related to adipocyte differentiation in subcutaneous abdominal fat obtained from insulin-sensitive (IS) and -resistant (IR) persons, matched for degree of moderate obesity. To determine whether other biological properties also differ between IR and IS obese individuals, we quantified markers of inflammatory activity in adipose tissue from overweight IR and IS individuals.Subcutaneous abdominal tissue was obtained from moderately obese women, divided into IR (n = 14) and IS (n = 19) subgroups by determining their steady-state plasma glucose (SSPG) concentrations during the insulin suppression test. Inflammatory activity was assessed by comparing expression of nine relevant genes and by immunohistochemical quantification of CD45- and CD68-containing cells.SSPG concentrations were approximately threefold higher in IR than in IS individuals. Expression levels of CD68, EMR1, IL8, IL6 and MCP/CCL2 mRNAs were modestly but significantly increased (p < 0.05) in IR compared with IS participants. Results of immunohistochemical staining were consistent with gene expression data, demonstrating modest differences between IR and IS individuals. Crown-like structures, in which macrophages surround single adipocytes, were rarely seen in tissue from either subgroup.A modest increase in inflammatory activity was seen in subcutaneous adipose tissue from IR compared with equally obese IS individuals. Together with previous evidence of impaired adipose cell differentiation in IR vs equally obese individuals, it appears that at least two biological processes in subcutaneous adipose tissue characterize the insulin-resistant state independent of obesity per se.

Abstract

Macrophages play important roles in the immunological defense system, but at the same time they are involved in inflammatory diseases such as atherosclerosis. Therefore, imaging macrophages is critical to assessing the status of these diseases. Toward this goal, a recombinant human H chain ferritin (rHFn)-iron oxide nano composite has been investigated as an MRI contrast agent for labeling macrophages. Iron oxide nanoparticles in the form of magnetite (or maghemite) with narrow size distribution were synthesized in the interior cavity of rHFn. The composite material exhibited the R(2) relaxivity comparable to known iron oxide MRI contrast agents. Furthermore, the mineralized protein cages are readily taken up by macrophages in vitro and provide significant T2* signal loss of the labeled cells. These results encourage further investigation into the development of the rHFn-iron oxide contrast agent to assess inflammatory disease status such as macrophage-rich atherosclerotic plaques in vivo.

Abstract

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.

Abstract

Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse.We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes.This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment.

Abstract

Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is increasingly recognized as a putative biomarker in cardiovascular and renal disease. Elevated plasma levels of ADMA are the consequence of increased synthesis, reduced renal clearance or reduced enzymatic degradation. Based upon the metabolic fate the highest plasma concentrations of ADMA have been reported in patients with renal failure in whom this molecule accumulates. However, the range of published ADMA levels in patients with chronic renal failure as well as in patients with end-stage renal failure undergoing maintenance hemodialysis, peritoneal dialysis or kidney transplant recipients is widely scattered and overlaps with the levels reported in healthy individuals. This wide distribution can in part be explained by different bioanalytical techniques and the lack of standardization of such assays. This review summarizes available literature on ADMA in patients with kidney disease and stresses the urgent need for a consensus regarding reference values for different analytical methods in order to appreciate the prognostic significance of elevated ADMA levels. At present, one cannot advocate this molecule for risk assessment or individual patient prognosis in the clinical work-up of patients with renal impairment.

Abstract

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.

Abstract

Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O(2) environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1alpha gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CoCl(2)) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl(2)-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1alpha-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia- and insulin-induced apelin expression.

Abstract

Serum inflammatory markers correlate with outcome and response to therapy in subjects with cardiovascular disease. However, current individual markers lack specificity for the diagnosis of coronary artery disease (CAD). We hypothesize that a multimarker proteomic approach measuring serum levels of vascular derived inflammatory biomarkers could reveal a "signature of disease" that can serve as a highly accurate method to assess for the presence of coronary atherosclerosis. We simultaneously measured serum levels of seven chemokines [CXCL10 (IP-10), CCL11 (eotaxin), CCL3 (MIP1 alpha), CCL2 (MCP1), CCL8 (MCP2), CCL7 (MCP3), and CCL13 (MCP4)] in 48 subjects with clinically significant CAD ("cases") and 44 controls from the ADVANCE Study. We applied three classification algorithms to identify the combination of variables that would best predict case-control status and assessed the diagnostic performance of these models with receiver operating characteristic (ROC) curves. The serum levels of six chemokines were significantly higher in cases compared with controls (P < 0.05). All three classification algorithms entered three chemokines in their final model, and only logistic regression selected clinical variables. Logistic regression produced the highest ROC of the three algorithms (AUC = 0.95; SE = 0.03), which was markedly better than the AUC for the logistic regression model of traditional risk factors of CAD without (AUC = 0.67; SE = 0.06) or with CRP (AUC = 0.68; SE = 0.06). A combination of serum levels of multiple chemokines identifies subjects with clinically significant atherosclerotic heart disease with a very high degree of accuracy. These results need to be replicated in larger cross-sectional studies and their prognostic value explored.

Abstract

The biological mechanism by which obesity predisposes to insulin resistance is unclear. One hypothesis is that larger adipose cells disturb metabolism via increased lipolysis. While studies have demonstrated that cell size increases in proportion to BMI, it has not been clearly shown that adipose cell size, independent of BMI, is associated with insulin resistance. The aim of this study was to test this widely held assumption by comparing adipose cell size distribution in 28 equally obese, otherwise healthy individuals who represented extreme ends of the spectrum of insulin sensitivity, as defined by the modified insulin suppression test.Subcutaneous periumbilical adipose tissue biopsy samples were fixed in osmium tetroxide and passed through the Beckman Coulter Multisizer to obtain cell size distributions. Insulin sensitivity was quantified by the modified insulin suppression test. Quantitative real-time PCR for adipose cell differentiation genes was performed for 11 subjects.All individuals exhibited a bimodal cell size distribution. Contrary to expectations, the mean diameter of the larger cells was not significantly different between the insulin-sensitive and insulin-resistant individuals. Moreover, insulin resistance was associated with a higher ratio of small to large cells (1.66 +/- 1.03 vs 0.94 +/- 0.50, p = 0.01). Similar cell size distributions were observed for isolated adipose cells. The real-time PCR results showed two- to threefold lower expression of genes encoding markers of adipose cell differentiation (peroxisome proliferator-activated receptor gamma1 [PPARgamma1], PPARgamma2, GLUT4, adiponectin, sterol receptor element binding protein 1c) in insulin-resistant compared with insulin-sensitive individuals.These results suggest that after controlling for obesity, insulin resistance is associated with an expanded population of small adipose cells and decreased expression of differentiation markers, suggesting that impairment in adipose cell differentiation may contribute to obesity-associated insulin resistance.

Abstract

Endothelial dysfunction by the loss of nitric oxide (NO) is a critical event during reperfusion of ischemic myocardium. Reduced NO availability signals important pathophysiological changes leading to myocardial reperfusion injury. We have recently shown that NO biosynthesis can be disturbed by the endogenous NO synthase (NOS) inhibitor ADMA and that these changes are mediated by an impairment of its metabolism by dimethylarginine dimethylaminohydrolase (DDAH). We therefore analyzed the role of ADMA and its metabolism in the setting of myocardial ischemia and reperfusion.C57-bl6 mice underwent myocardial ischemia for exactly 30 min followed by 2, 4, 8, 12, 24, and 72 h of reperfusion achieved by occlusion and re-opening of the left coronary artery. The reperfused left ventricle was subsequently homogenized for measurements of determinants of the NO synthase pathway. Furthermore, the effects and its mechanisms of ADMA on reperfusion injury were analyzed in a genetic mouse model.A significant accumulation of ADMA was found in myocardial tissue when mice were subjected to 30 min of ischemia followed by reperfusion in our in vivo model. The maximum increase of tissue ADMA at 4 h of reperfusion coincided with reductions of NO tissue concentrations and DDAH activity; protein expression of NOS isoforms, however, was not changed. Furthermore, DDAH overexpression in a genetic mouse model as well as treatment with oral L-arginine markedly reduced reperfusion injury by 40-50% at 4 h of reperfusion. The effects of ADMA on reperfusion injury were shown to be mediated by reduced eNOS activity and phosphorylation, expression of adhesion molecules, and leukocyte activity.Accumulation of tissue ADMA by impairment of DDAH was found to be a significant determinant of reperfusion injury. Our results indicate that ADMA could be a potential new target for the treatment of myocardial ischemia/reperfusion injury.

Abstract

Smoking is closely associated with insulin resistance, though the mechanism is not clear. Adiponectin, a novel anti-atherosclerotic and anti-inflammatory adipose tissue product, which is closely associated with insulin resistance, was reported to be low in smokers with cofactors for atherosclerosis. However, the effects of smoking on circulating adiponectin levels in otherwise healthy people are unknown. In this study, a case control design was implemented to search for the effect of smoking on plasma adiponectin and insulin sensitivities in healthy people. Sixty-four healthy male smokers, with no family history of hypertension and diabetes mellitus were compared with appropriate 36 age and body mass index matched controls. Both the patients and controls were the soldiers of a troop with regular daily physical activity. Plasma adiponectin, high sensitive C-reactive protein (hsCRP), insulin and lipid levels, and insulin sensitivity as assessed by homeostasis model assessment index (HOMA) of the smokers were measured and compared with those of the controls. The plasma adiponectin, hsCRP, insulin levels and HOMA indexes of the two groups were similar. These parameters were not affected by the amount of cigarettes per day. HDL-cholesterol levels were lower (p = 0.01) and systolic blood pressures were higher (p = 0.02) in the smokers. These results indicate that smoking may not affect plasma adiponectin and insulin levels in young and healthy men with high exercise capacity.

Abstract

Localization of atherosclerotic lesions in the abdominal aorta has been previously correlated to areas of adverse hemodynamic conditions, such as flow recirculation, low mean wall shear stress, and high temporal oscillations in shear. Along with its many systemic benefits, exercise is also proposed to have local benefits in the vasculature via the alteration of these regional flow patterns. In this work, subject-specific models of the human abdominal aorta were constructed from magnetic resonance angiograms of five young, healthy subjects, and computer simulations were performed under resting and exercise (50% increase in resting heart rate) pulsatile flow conditions. Velocity fields and spatial variations in mean wall shear stress (WSS) and oscillatory shear index (OSI) are presented. When averaged over all subjects, WSS increased from 4.8 +/- 0.6 to 31.6 +/- 5.7 dyn/cm2 and OSI decreased from 0.22 +/- 0.03 to 0.03 +/- 0.02 in the infrarenal aorta between rest and exercise. WSS significantly increased, whereas OSI decreased between rest and exercise at the supraceliac, infrarenal, and suprabifurcation levels, and significant differences in WSS were found between anterior and posterior sections. These results support the hypothesis that exercise provides localized benefits to the cardiovascular system through acute mechanical stimuli that trigger longer-term biological processes leading to protection against the development or progression of atherosclerosis.

Abstract

Plasma asymmetric dimethylarginine (ADMA) concentrations are higher in apparently healthy, insulin-resistant (IR) individuals and decrease in response to thiazolidenedione treatment.The objective of the study was to determine whether ADMA concentrations would also fall when insulin sensitivity is enhanced with weight loss in obese individuals. DESIGN/SETTING/PATIENTS/INTERVENTION: Twenty obese women classified as IR or insulin sensitive (IS) on the basis of their steady-state plasma glucose (SSPG) concentration during the insulin suppression test underwent 12 wk of dietary weight loss.Plasma glucose, insulin, and ADMA were measured at baseline and after weight loss; change in insulin resistance was quantified by repeating the SSPG after the dietary intervention.Although weight loss was similar in the two groups, significant improvements in SSPG, glucose, and insulin concentrations were confined to the IR group. Baseline plasma ADMA concentrations (mean +/- sd) were higher in IR subjects (1.69 +/- 0.44 vs. 1.18 +/- 0.45 micromol/liter, P = 0.02) and decreased to 1.20 +/- 0.22 micromol/liter (P < 0.001) with weight loss. In contrast, ADMA levels did not change with a similar extent of weight loss in the IS group.Plasma ADMA levels are higher in obese, IR women than in equally obese, IS women and decrease in response to weight loss when associated with enhancement of insulin sensitivity.

Abstract

At a population level, inflammatory markers have been shown to predict outcome and response to therapy in patients with atherosclerotic cardiovascular disease. However, current markers are not sufficiently sensitive or specific to provide clinical utility for managing individual patients. We hypothesize that measurement of multiple circulating disease-related inflammatory factors will be more informative, allowing the early identification of vascular wall disease activity. We have investigated whether protein microarray-based abundance measurements of circulating proteins can predict the severity of atherosclerotic disease. Using a longitudinal experimental design with apolipoprotein E-deficient mice and control C57Bl/6J and C3H/HeJ wild-type mice, we measured the time-related serum protein expression of 30 inflammatory markers using a protein microarray. We were able to identify a subset of proteins that classify and predict the severity of atherosclerotic disease with a high level of accuracy. The time-specific vascular expression of these markers was verified by showing that their gene expression in the mouse aorta correlated closely to the temporal pattern of serum protein levels. In conclusion, these data suggest that quantification of multiple disease-related inflammatory proteins can provide a more sensitive and specific methodology for assessing atherosclerotic disease activity in humans, and identify candidate biomarkers for such studies.

Abstract

THR0921 is a novel peroxisome proliferator-activated receptor gamma (PPARgamma) agonist with potent anti-diabetic properties. Because of the proposed role of PPARgamma in inflammation, we investigated the potential of orally active THR0921 to inhibit the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by the injection of bovine type II collagen in complete Freund's adjuvant on days 0 and 21. Mice were treated with THR0921 (50 mg/kg/day) starting on the day of the booster injection and throughout the remaining study period. Both clinical disease activity scores as well as histological scores of joint destruction were significantly reduced in mice treated with THR0921 compared to untreated mice. Proliferation of isolated spleen cells, as well as circulating levels of IgG antibody to type II collagen, was decreased by THR0921. Moreover, spleen cell production of IFN-gamma, tumor necrosis factor (TNF)-alpha and IL-1beta in response to exposure to lipopolysaccharide or type II collagen was reduced by in vivo treatment with THR0921. Steady state mRNA levels of TNF-alpha, IL-1beta, monocyte chemotactic protein-1 and receptor activator of nuclear factor kappaB ligand (RANKL) in isolated joints were all decreased in mice treated with THR0921. Finally, THR0921 inhibited osteoclast differentiation of bone marrow-derived cells stimulated with macrophage colony-stimulating factor and RANKL. In conclusion, THR0921 attenuates collagen-induced arthritis in part by reducing the immune response. As such, PPARgamma may be an important therapeutic target for rheumatoid arthritis.

Abstract

Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease.

Abstract

Caveolae (sphingolipid- and cholesterol-rich, 100 nm flask-shaped invaginations of the cell membrane) serve as a nexus of cell signalling. In the present study caveolin-rich lipid raft domains were extracted from HUVEC (human umbilical-vein endothelial cells) using both density gradient and immunoprecipitation techniques, and demonstrated localization of the TGF-beta (transforming growth factor-beta) receptors TbetaRI and TbetaRII to the Cav-1 (caveolin-1)-enriched raft fractions of these normal, human endothelial cells. Immunoprecipitation demonstrated an association between TbetaRI and TbetaRII, as well as an association of the TbetaRs receptors with Cav-1 and eNOS (endothelial nitric oxide synthase), suggesting a mutual co-localization to caveolae; after treatment of HUVEC with 5 ng/ml TGF-beta1 for 15 min, however, co-precipitation of eNOS with TbetaRI, TbetaRII and Cav-1 was diminished. The loss of immunoprecipitable eNOS from Cav-1-enriched fractions was accompanied by a decrease both in phosphorylation of eNOS and in enzymatic activity (conversion of arginine into citrulline). No change in the localization of eNOS to morphologically distinct caveolae could be detected by electron microscopy after treatment of HUVEC with TGF-beta1 for 20 min. The results of these investigations provide evidence that TbetaRI interacts with eNOS in the caveolae of normal, human endothelial cells and has a regulatory function on basal eNOS enzymatic activity.

Abstract

The propensity for developing atherosclerosis is dependent on underlying genetic risk and varies as a function of age and exposure to environmental risk factors. Employing three mouse models with different disease susceptibility, two diets, and a longitudinal experimental design, it was possible to manipulate each of these factors to focus analysis on genes most likely to have a specific disease-related function. To identify differences in longitudinal gene expression patterns of atherosclerosis, we have developed and employed a statistical algorithm that relies on generalized regression and permutation analysis. Comprehensive annotation of the array with ontology and pathway terms has allowed rigorous identification of molecular and biological processes that underlie disease pathophysiology. The repertoire of atherosclerosis-related immunomodulatory genes has been extended, and additional fundamental pathways have been identified. This highly disease-specific group of mouse genes was combined with an extensive human coronary artery data set to identify a shared group of genes differentially regulated among atherosclerotic tissues from different species and different vascular beds. A small core subset of these differentially regulated genes was sufficient to accurately classify various stages of the disease in mouse. The same gene subset was also found to accurately classify human coronary lesion severity. In addition, this classifier gene set was able to distinguish with high accuracy atherectomy specimens from native coronary artery disease vs. those collected from in-stent restenosis lesions, thus identifying molecular differences between these two processes. These studies significantly focus efforts aimed at identifying central gene regulatory pathways that mediate atherosclerotic disease, and the identification of classification gene sets offers unique insights into potential diagnostic and therapeutic strategies in atherosclerotic disease.

Abstract

One of the earliest observable events in atherogenesis is enhanced monocyte adhesion to the endothelium. In addition to reducing circulating levels of cholesterol, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) are thought to have direct salutary effects upon vascular cells. We hypothesized that the new statin, rosuvastatin, would have anti-inflammatory effects on the vessel wall. Eight-week-old apolipoprotein E-deficient mice were fed a normal chow diet for a period of 12 weeks. During this time mice were administered vehicle or rosuvastatin at a dose of 0, 1, 5, or 20 mg/kg by subcutaneous injection at the same time daily for a period of 2 or 6 weeks prior to sacrifice. At the end of the study, rosuvastatin-treated animals displayed lower plasma total cholesterol levels, whereas showing little change in high-density lipoprotein cholesterol or triglycerides. Using a functional binding assay, we also demonstrated that endothelial adhesiveness for monocytes was significantly attenuated after 2 weeks of treatment with rosuvastatin. Quantitative real-time polymerase chain reaction determined that rosuvastatin reduced the expression of vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and metalloproteinase-9 in the vessel wall. In addition, rosuvastatin inhibited vascular expression of p22(phox) and superoxide production, as well as diminishing plasma 8-isoprostanes concentrations. Thus, treatment with rosuvastatin has acute anti-inflammatory actions that likely participate in its beneficial actions during atherogenesis.

Abstract

Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice.We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis.Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study. To gain insights into the molecular pathways that are differentially activated in strains of mice with varied susceptibility to atherosclerosis, we performed comprehensive transcriptional profiling of their vascular wall. Genes identified through these studies expand the repertoire of factors in disease-related signaling pathways and identify novel candidate genes in atherosclerosis.

Abstract

Thiazolidinedione (TZD) compounds enhance insulin sensitivity and attenuate inflammation. The effect of the TZD compound, rosiglitazone (RSG) on both actions was evaluated in two groups of insulin-resistant subjects with minimal elevations of fasting plasma glucose (PG) concentration: group A (n=15, PG < 7.0 mmol/L) and group B (n=14, PG 7.0-8.3 mmol/L). Insulin action, quantified by the insulin suppression test, improved after three months of treatment in both groups, and concentrations of C-reactive protein, plasminogen activator inhibitor-1 and Eselectin all fell. Significant decreases in L-selectin and P-selectin were confined to group B, and concentrations of interleukin-6, intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 did not fall in either group. Significant relationships were not discerned between enhanced insulin sensitivity and related variables and decreases in inflammatory/vascular markers, suggesting that RSG-induced changes in the latter variables in insulin-resistant individuals might be at least partly independent of the effects of the drug on insulin action.

Abstract

After cardiac transplantation, graft damage occurs secondary to ischemia-reperfusion injury and acute rejection. This damage ultimately leads to the development of graft coronary artery disease (GCAD), which limits long-term graft survival. Apoptosis is directly involved in graft injury, contributing to the development of GCAD. To assess the role of the antiapoptotic factor Bcl-2 in the process of GCAD, we transplanted hearts from FVB transgenic mice overexpressing human Bcl-2 under the control of alpha-myosin heavy chain promoter into allogenic C57BL/6 mice. Bcl-2 overexpression led to reduced cytochrome c-mediated caspase-9-dependent cardiomyocyte apoptosis and local inflammation (neutrophil infiltration and proinflammatory cytokine production) in cardiac allografts during ischemia-reperfusion injury and also led to reduced immune responses (inflammatory cell infiltration, production of T(H)1 cytokines and chemokines, and expression of adhesion molecules) during acute and chronic rejection without affecting host CD4(+) and CD8(+) cell responses in the spleen. Thus, local Bcl-2 expression directly contributes to the modulation of local immune responses in allograft rejection, resulting in attenuated GCAD. In conclusion, our findings suggest that the modulation of Bcl-2 expression by pharmacologic up-regulation or gene transfer may be of clinical benefit in the short- and long-term function of cardiac allografts.

Abstract

Mesodermal and epidermal precursor cells undergo phenotypic changes during differentiation to the smooth muscle cell (SMC) lineage that are relevant to pathophysiological processes in the adult. Molecular mechanisms that underlie lineage determination and terminal differentiation of this cell type have received much attention, but the genetic program that regulates these processes has not been fully defined. Study of SMC differentiation has been facilitated by development of the P19-derived A404 embryonal cell line, which differentiates toward this lineage in the presence of retinoic acid and allows selection for cells adopting a SMC fate through a differentiation-specific drug marker. We sought to define global alterations in gene expression by studying A404 cells during SMC differentiation with oligonucleotide microarray transcriptional profiling. Using an in situ 60-mer array platform with more than 20,000 mouse genes derived from the National Institute on Aging clone set, we identified 2,739 genes that were significantly upregulated after differentiation was completed (false-detection ratio <1). These genes encode numerous markers known to characterize differentiated SMC, as well as many unknown factors. We further characterized the sequential patterns of gene expression during the differentiation time course, particularly for known transcription factor families, providing new insights into the regulation of the differentiation process. Changes in genes associated with specific biological ontology-based pathways were evaluated, and temporal trends were identified for functional pathways. In addition to confirming the utility of the A404 model, our data provide a large-scale perspective of gene regulation during SMC differentiation.

Abstract

Ischemia-reperfusion injury is an important risk factor for graft coronary artery disease (GCAD). We hypothesized that overexpression of SOD1 in donor hearts would suppress ischemia-reperfusion injury and thereby reduce GCAD.In one series, donor hearts of C57BL/6 (H-2b) transgenic mice overexpressing human SOD1 or C57BL/6 wild-type mice were heterotopically transplanted into C57BL/6 recipients and procured after 4 hours of reperfusion (n=6 each). Superoxide, TNF-alpha, and MCP-1/CCL2 production were significantly reduced in the SOD1 transgenic donor heart recipients, and graft injury determined by serum CPK-MB levels was significantly decreased. Cardiomyocyte apoptosis and caspase-3 and caspase-9 activities were significantly decreased in these recipients; caspase-8 activity was unchanged. Fas ligand but not Fas expression was also reduced. In a second series, transgenic and wild-type hearts were transplanted into C-H-2bm12KhEg (H-2bm12) recipients, and then procured on day 56 (n=7 each). Cardiac graft beating was significantly better in the SOD1 transgenic donor heart recipients on days 28, 42, and 56 (but not day 14). Significant reduction in luminal narrowing, the intima/media ratio, and the percentage of diseased vessels was seen in the SOD1 transgenic donor heart recipients, and MCP-1/CCL2, ICAM-1, and VCAM-1 production were significantly reduced.Overexpression of SOD1 attenuates both apoptosis and the inflammatory response during ischemia-reperfusion injury and therefore mitigates against the subsequent development of GCAD.

Abstract

Stent-based delivery of sirolimus (SRL) has shown reduction in neointimal hyperplasia and restenosis. The purpose of this study was to evaluate the chronic vascular response and the expression of cell cycle regulators after SRL-eluting stent implantation in a porcine coronary model.Forty-nine pigs underwent placement of 109 oversized stents (control, n=54, SRL (140 microg/cm(2)), n=55) in the coronary arteries with histologic analysis and Western blot (PCNA, p27(kip1), CD45, MCP-1, IL-2, IL-6, TNF-beta) at 3, 30, 90 or 180 days.At 3 days, the mean thrombus area was similar for control (0.38+/-0.19 mm(2)) and SRL (0.29+/-0.09 mm(2)) stents. After 30 days, the mean neointimal area was significantly less for the SRL (1.40+/-0.35 mm(2)) versus the control stents (2.94+/-1.28 mm(2), p<0.001). At 90 and 180 days, the mean neointimal area was similar for the SRL (3.03+/-0.92 and 3.34+/-0.99 mm(2)) as compared with control stents (3.45+/-1.09 and 3.65+/-1.23 mm(2)). Western blot analysis demonstrated an increased expression of p27(kip1) in the vessel wall at 90 days for the SRL versus control stents (p=0.05) but with increased levels of PCNA in the SRL as compared with control stents (p=0.003).SRL-eluting stents favorably modulate neointimal formation for 30 days in the porcine coronary model. Long-term inhibition of neointimal hyperplasia is not sustained presumably due to delayed cellular proliferation despite increased levels of the cyclin-dependent kinase p27(kip1) in the vessel wall.

Abstract

NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility. Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis.We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed approximately 2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (P<0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production.DDAH I overexpression increases NOS activity in vitro and in vivo. The hDDAH-1 transgenic animal exhibits a reduced systolic blood pressure, systemic vascular resistance, and cardiac stroke volume. This study provides compelling evidence that the elaboration and metabolism of endogenous ADMA plays an important role in regulation of NOS activity.

Abstract

Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin-angiotensin receptor-like 1 (APJ) signaling has ever been described.We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein-coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction.The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.

Abstract

A growing body of evidence has demonstrated that oxidants play a critical role in the pathogenesis of endothelial dysfunction. Pathologic processes fundamental to development and progression of endothelial dysfunction such as the oxidation of LDL, the loss of bioavailable nitric oxide, and the vascular inflammatory response are all modulated by oxidant stress. Therapeutic strategies to reverse endothelial dysfunction have begun to focus on agents with the ability to ameliorate oxidant stress.Preclinical and clinical studies evaluating the actions of antioxidants as well as traditional cardiovascular therapies in ameliorating oxidative stress and endothelial dysfunction were reviewed through the use of a MEDLINE search of English language articles published between the years of 1992 and 2002.Antioxidants appear to be an attractive candidate therapy, yet despite compelling preclinical evidence supporting their benefits, efforts to validate the use of vitamins C and E in a clinical setting have been conflicting. In contrast, conventional cardiovascular therapies such as ACE inhibitors, statins, insulin-sensitizing agents, and estrogens have been shown to alleviate endothelial dysfunction, often independent of their effects on systemic disease processes.These agents restore endothelial function through their salutary effects on pathologic vascular oxidative processes.

Abstract

Vascular endothelial cells maintain the interface between the systemic circulation and soft tissues and mediate critical processes such as inflammation in a vascular bed-selective fashion. To expand our understanding of the genetic pathways that underlie these specific functions, we have focused on the identification of novel genes that are differentially expressed in all endothelial cells, as well as restricted groups of this cell type. Virtual subtraction was conducted employing gene expression data deposited in public databases and 384 genes identified. These genes were spotted on custom microarrays, along with 288 genes identified through subtraction cloning from TGF-beta-stimulated endothelial cells. Arrays were evaluated with RNA samples representing endothelial cells cultured from four vascular sources and five non-endothelial cell types. These studies identified 64 pan-endothelial markers that were differentially expressed with at least a threefold difference (range 3- to 55-fold). In addition, differences in gene expression profiles among endothelial cells from different vascular beds were identified. Validation of these findings was performed by RNA blot expression studies, and a number of the novel genes were shown to be expressed under angiogenic conditions in the developing mouse embryo. The combined tools of database mining and transcriptional profiling thus provide expanded knowledge of endothelial cell gene expression and endothelial cell biology.

Insulin resistance and compensatory hyperinsulinemia - The key player between cigarette smoking and cardiovascular disease?JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGYReaven, G., Tsao, P. S.2003; 41 (6): 1044-1047

Abstract

Hyperinsulinemia, dyslipidemia, and endothelial dysfunction are characteristic findings in insulin-resistant individuals, and all of these abnormalities have been identified as increasing cardiovascular disease (CVD) risk. Smokers tend to be relatively insulin resistant, hyperinsulinemic, and dyslipidemic, with evidence of endothelial dysfunction, as compared with nonsmokers, and recent epidemiologic data have suggested that CVD in smokers is primarily seen in those individuals who also have the characteristic findings of insulin resistance. Based on these observations, it is argued that insulin resistance and its consequences represent a major mechanistic link between cigarette smoking and CVD. It is also postulated that the enhanced CVD risk in smokers, resulting from hyperinsulinemia, abnormalities of lipoprotein metabolism, and endothelial dysfunction, will primarily be present in those smokers who are insulin resistant. As a corollary, it is suggested that CVD risk in individuals who cannot, or will not, stop smoking can be reduced by therapeutic efforts aimed at attenuating the adverse effects of insulin resistance and its consequences.

Abstract

The present study was designed to determine the efficiency of translocation of short polymers of arginine into vascular smooth muscle cells (VSMC) and to determine their effect on nitric oxide (NO) synthesis. Immunostaining revealed that heptamers of L-arginine (R7) rapidly translocated into the VSMC. This rapid transport was not observed with shorter polymers of L-arginine (R5) nor heptamers of lysine (K7). Translocation of R7 was not inhibited by the addition of free L-arginine into the media. When cells were transiently pretreated with R7 or a nonamer of arginine (R9), NO(2) production from cytokine stimulated VSMC was significantly increased, whereas incubation with R5 and K7 had no effect. Short polymers of arginine not only have a unique ability of rapid VSMC translocation but once internalized enhance NO production. Heptamers (or larger polypeptides) of arginine may be useful in therapy to enhance NO production in the vascular system.

Abstract

This study was initiated to see if plasma asymmetric dimethylarginine (ADMA) concentrations decreased in hyperglycemic patients with type 2 diabetes following metformin treatment, either as monotherapy or following its addition to sulfonylurea-treated patients. Fasting plasma glucose, dimethylarginine, and L-arginine concentrations were measured before and 3 months after the administration of a maximally effective dose of metformin to 31 patients with type 2 diabetes in poor glycemic control (fasting plasma concentrations > 9.7 mmol/L), while being treated with either diet (n = 16) or a maximal amount of a sulfonylurea compound (n = 15). Fasting plasma glucose concentration (mean +/- SEM) decreased to a similar degree (P

Abstract

Increased levels of asymmetric dimethylarginine (ADMA) are associated with endothelial dysfunction and increased risk of cardiovascular disease. Several cardiovascular risk factors are associated with reduced sensitivity to insulin, but elevated ADMA concentrations have not been fully linked to the metabolic syndrome.To evaluate the relationship between insulin sensitivity and plasma ADMA concentrations, and to determine whether a pharmacological treatment that increases insulin sensitivity would also modulate ADMA concentrations.Cross-sectional study, containing a nonrandomized controlled trial component, of 64 healthy volunteers without diabetes (42 women, 22 men; 48 with normal blood pressure and 16 with hypertension), which was conducted at a university medical center between October 2000 and July 2001.Rosiglitazone (4 mg/d for 4 weeks and then 4 mg twice daily for 8 weeks), an insulin-sensitizing agent, was given to 7 insulin-resistant subjects with hypertension. These subjects were studied before and after 12-week treatment.Insulin sensitivity measured by the insulin suppression test, and fasting plasma levels of low-density lipoprotein cholesterol, triglycerides, high-density lipoprotein cholesterol, glucose, insulin, and ADMA concentrations.Plasma ADMA concentrations were positively correlated with impairment of insulin-mediated glucose disposal in nondiabetic, normotensive subjects (r = 0.73; P

Abstract

Hyperhomocysteinemia is a putative risk factor for cardiovascular disease, which also impairs endothelium-dependent vasodilatation. A number of other risk factors for cardiovascular disease may exert their adverse vascular effects in part by elevating plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Accordingly, we determined if homocysteine could increase ADMA levels.When endothelial or nonvascular cells were exposed to DL-homocysteine or to its precursor L-methionine, ADMA concentration in the cell culture medium increased in a dose- and time-dependent fashion. This effect was associated with the reduced activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Furthermore, homocysteine-induced accumulation of ADMA was associated with reduced nitric oxide synthesis by endothelial cells and segments of pig aorta. The antioxidant pyrrollidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation. Moreover, homocysteine dose-dependently reduced the activity of recombinant human DDAH in a cell free system, an effect that was due to a direct interaction between homocysteine and DDAH.Homocysteine post-translationally inhibits DDAH enzyme activity, causing ADMA to accumulate and inhibit nitric oxide synthesis. This may explain the known effect of homocysteine to impair endothelium-mediated nitric oxide-dependent vasodilatation.

Abstract

Advanced age is associated with endothelial dysfunction and increased risk for atherosclerosis. However, the mechanisms for these observed effects are not clear. To clarify the association between aging and loss of endothelial function, young human aortic endothelial cells (HAECs), senescent HAECs transfected with control vector, and immortalized HAECs containing human telomerase reverse transcriptase (hTERT) were compared for expression of endothelial nitric oxide synthase (eNOS) and production of NO. To investigate a specific function modulated by endothelial NO, adhesion of monocytes under basal conditions as well as after exposure to TNF-alpha was assessed. A decrease in eNOS mRNA, protein, and activity was observed in endothelial cells at senescence as compared with young HAEC; this effect was blunted in hTERT cells. In all cells, shear stress induced a greater increase in the expression of eNOS protein with the final result being higher levels in hTERT compared with senescent cells. Basal monocyte binding was significantly elevated on aged endothelial cells compared with parental and hTERT cells. Exposure of TNF-alpha resulted in a 2-fold increase in monocyte adhesion in senescent cells, whereas this effect was reduced in cells transfected with hTERT. Prior exposure to fluid flow significantly reduced subsequent monocyte adhesion in all groups. These studies demonstrate that replicative aging results in decreased endothelial expression of eNOS accompanied by enhanced monocyte binding. Stable expression of hTERT results in endothelial cells with a younger phenotype with greater amount of eNOS and NO activity. Thus, telomerase transfection may have important functional consequences on endothelial cells.

Abstract

We provide anatomic and functional evidence that nicotine induces angiogenesis. We also show that nicotine accelerates the growth of tumor and atheroma in association with increased neovascularization. Nicotine increased endothelial-cell growth and tube formation in vitro, and accelerated fibrovascular growth in vivo. In a mouse model of hind-limb ischemia, nicotine increased capillary and collateral growth, and enhanced tissue perfusion. In mouse models of lung cancer and atherosclerosis, we found that nicotine enhanced lesion growth in association with an increase in lesion vascularity. These effects of nicotine were mediated through nicotinic acetylcholine receptors at nicotine concentrations that are pathophysiologically relevant. The endothelial production of nitric oxide, prostacyclin and vascular endothelial growth factor might have a role in these effects.

Abstract

Diabetes mellitus (DM) is a primary risk factor for cardiovascular disease. Although recent studies have demonstrated an important role for extracellular matrix metalloproteinases (MMPs) in atherosclerosis, little is known about the effects of hyperglycemia on MMP regulation in vascular cells. Gelatin zymography and Western blot analysis revealed that the activity and expression of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2) gelatinase, were significantly increased in vascular tissue and plasma of two distinct rodent models of DM. Bovine aortic endothelial cells (BAECs) grown in culture did not express MMP-9 constitutively; however, chronic (2-week) incubation with high glucose medium induced MMP-9 promoter activity, mRNA and protein expression, and gelatinase activity in BAECs. On the other hand, high glucose culture did not change MMP-9 activity from vascular smooth muscle cells or macrophages. Electron paramagnetic resonance studies indicate that BAECs chronically grown in high glucose conditions produce 70% more ROS than do control cells. Enhanced MMP-9 activity was significantly reduced by treatment with the antioxidants polyethylene glycol-superoxide dismutase and N-acetyl-L-cysteine but not by inhibitors of protein kinase C. In conclusion, vascular MMP-9 activity is increased in DM, in part because of enhanced elaboration from vascular endothelial cells, and oxidative stress plays an important role. This novel mechanism of redox-sensitive MMP-9 expression by hyperglycemia may provide a rationale for antioxidant therapy to modulate diabetic vascular complications.

Abstract

-The predominant cause of restenosis after angioplasty is now thought to be inward remodeling, but the mechanisms responsible are unknown. Remodeling in normal vessels is regulated by the endothelium in response to altered shear stress. Although the endothelium is often damaged by angioplasty, restenosis rates after angioplasty have been correlated with impaired coronary flow. Thus, we examined how increases or decreases in blood flow through balloon catheter-injured rat carotid arteries affect vessel morphometry (4, 10, and 28 days), cell migration (4 days), and levels of promigratory mRNAs (2 and 10 days). After 28 days, the luminal area in vessels with low blood flow was significantly less than in those with normal and high blood flow (0.17+/-0.01 [low] versus 0.24+/-0.06 [normal] versus 0.30+/-0.02 [high] mm(2), P:<0.01), predominantly because of accentuated inward remodeling (or reduced area within the external elastic lamina; 0.42+/-0.02 [low] versus 0.54+/-0.07 [normal] versus 0.53+/-0.04 [high] mm(2), P:<0.05). Low flow also enhanced smooth muscle cell migration 4 days after injury by 90% above normal and high flows (P:<0.01). Two days after injury, low flow significantly increased levels of mRNAs encoding promigratory peptides (integrin alpha(v)ss(3), transforming growth factor-ss(1), CD44v6, MDC9, urokinase plasminogen activator receptor, and ss-inducible gene h3); these changes persisted 10 days after injury and were localized to the neointima. Low blood flow may promote restenosis after angioplasty because of its adverse effect on vessel remodeling, and it is associated with the augmented expression of multiple genes central to cell migration and restenosis.

Abstract

Vascular endothelial cells are constantly subjected to pressure-induced cyclic strain. Reactive oxygen species (ROS) have been implicated in atherosclerosis and vascular remodeling. Recent evidence indicates that a vascular NAD(P)H oxidase may be an important source of ROS in both physiologic and pathophysiologic situations. The aim of this study was to investigate cyclic strain-induced NAD(P)H oxidase activity in endothelial cells. ROS production was examined by electron paramagnetic resonance and lucigenin chemiluminescence. Cyclic strain-induced NAD(P)H oxidase activity was quantified by activity assay while the expression of p22phox was monitored by Northern blotting. Endothelial cells produce basal amounts of ROS that were enhanced by cyclic strain. Moreover subsequent stimulation with TNF-alpha resulted in significantly greater ROS production in cells previously exposed to cyclic strain as compared to static conditions. Cyclic strain resulted in a significant increase in message for the p22phox subunit as well as activity of the NAD(P)H oxidase. The induced oxidative stress was accompanied by increased mobilization of the transcription factor NFkappaB, an effect that was blocked by a pharmacological inhibitor of NAD(P)H. These results demonstrate a pivotal role for NAD(P)H oxidase in cyclic strain-induced endothelial ROS production and may provide insight into the modulation of vascular disease by biomechanical forces. J. Cell. Biochem. Suppl. 36: 99-106, 2001.

Abstract

Atherosclerotic lesions display a nonuniform distribution throughout the vascular tree. Mechanical forces produced by local alterations in blood flow may play an important role in the localization of atherosclerosis. One such force, cyclic strain, has been hypothesized to promote atherogenesis by inducing oxidative stress in endothelial cells, resulting in enhanced endothelial adhesiveness for monocytes. To investigate the signal transduction systems involved, human aortic endothelial cells were plated on flexible silicone strips that were either non-coated or adsorbed with poly-L-lysine, vitronectin, fibronectin, or collagen I. Cells were then subjected to uniform sinusoidal stretch (10%) for 6 h. Endothelial superoxide anion production was increased in cells exposed to cyclic strain compared to static conditions. Furthermore, endothelial oxidative response to stretch was matrix protein-dependent, whereas cells grown on fibronectin and collagen I produced significantly more superoxide. The oxidative response to cyclic strain was reduced by coincubation with RGD peptides, blocking antibodies to alpha2- and beta-integrins antibodies, as well as inhibitors of protein kinase C. To investigate the effect of oxidative stress on gene transcription, endothelial cells grown on collagen I were transfected with an NFkappaB-sensitive luciferase construct. Cells that underwent cyclic strain displayed a tenfold induction of NFkappaB activation compared to static controls. Strain-induced luciferase activity was blunted by coincubation with RGD peptides or calphostin C. Thus, exposure of endothelial cells to cyclic strain led to integrin activation of a PKC-sensitive pathway that results in increased superoxide anion production and mobilization of NFkappaB.

Abstract

We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes.Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase.Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2-) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-KB by electromobility gel shift assay.Incubation of endothelial cells with ADMA (0.1 microM to 100 microM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 microM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2- to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-KB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody.We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

Abstract

A lipoprotein lipase-like gene was recently cloned from endothelial cells. In vitro functional experiments have suggested that this endothelial-derived lipase (EDL) has phospholipase activity, and preliminary in vivo studies have suggested a role in the regulation of high-density lipoprotein metabolism. To investigate local control of lipase activity and lipid metabolism in the blood vessel wall, we have examined the regulation of EDL expression in cultured human umbilical vein and coronary artery endothelial cells. EDL mRNA levels were upregulated in both cell types by inflammatory cytokines implicated in vascular disease etiology, including TNF-alpha and IL-1beta. In addition, both fluid shear stress and cyclic stretch were found to increase the EDL mRNA levels in these cultured cells. This highly regulated expression of EDL in vascular endothelial cells suggests that this recently identified lipase is intricately involved in modulating vessel wall lipid metabolism and may play a role in vascular diseases such as atherosclerosis.

Abstract

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is elevated in hypercholesterolemia. This study was designed to determine the role of ADMA in the increased mononuclear cell adhesiveness observed in human hypercholesterolemia. In patient studies, plasma ADMA levels were determined by high-performance liquid chromatography. Functional mononuclear leukocyte adhesion assays were performed in parallel, and flow cytometry was used to characterize bound monocytes and T lymphocytes. Hypercholesterolemic patients were then placed on an oral L-arginine regimen of 14 or 21 g/d and studied over 12 weeks. In cell culture studies, bovine aortic endothelial cells were incubated with varied concentrations of ADMA. Monocytoid cells were cocultured with these bovine aortic endothelial cells, and their adhesiveness was assessed by use of a binding assay. Flow cytometry was used to quantify adhesion molecule expression. Plasma ADMA levels and adhesiveness of mononuclear cells (specifically, monocytes and T lymphocytes) were elevated in hypercholesterolemic patients. Adhesiveness was inversely correlated with the plasma L-arginine/ADMA ratio. Oral administration of L-arginine normalized plasma L-arginine/ADMA ratios and attenuated monocyte and T-lymphocyte adhesiveness. ADMA had no direct effect on the adhesiveness of mononuclear cells. However, monocytes became hyperadhesive when cocultured with ADMA-exposed endothelial cells. In human hypercholesterolemia, the plasma L-arginine/ADMA ratio is inversely correlated with mononuclear cell adhesiveness. Restoration of the L-arginine/ADMA ratio to control levels normalizes mononuclear cell adhesiveness. Our studies suggest that the elaboration of endothelium-derived nitric oxide affects the behavior of circulating T lymphocytes and monocytes.

Abstract

The purpose of the study was to investigate the role of nitric oxide (NO) in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA.Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC.Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence.The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by +60% (n = 6), which increased further when EC were stimulated by shear stress (24 h) by +137% (n = 5) as compared with untransfected, unstimulated EC (both p < 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r = -0.5669, p < 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha.Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation.

Abstract

To extend our previous observation that thoracic aortae from rats with spontaneous hypertension (SHR) bind monocytoid cells with enhanced avidity, we isolated thoracic aortae from two different forms of rodent hypertension: Dahl salt-sensitive (Dahl-S) rats fed a high salt diet and Sprague-Dawley (S-D) rats fed a fructose-enriched diet. Blood pressure was determined 14 days after feeding normal chow or chow containing 8% NaCl to Dahl-S and Dahl salt-resistant (Dahl-R) rats, and either chow or a fructose-enriched diet to S-D and Fischer 344 (F-344) rats. Blood pressure was similar in Dahl-S and Dahl-R rats on the chow diet, but higher in Dahl-S rats in response to the 8% NaCl diet (188 +/- 7 v 137 +/- 3 mm Hg, P < .001). Blood pressure also increased when S-D rats consumed fructose as compared with chow (149 +/- 4 v 128 +/-2, P < .05), whereas blood pressure did not change with diet in F-344. Thoracic aortae were removed from rats in each experimental group, and their ability to bind murine monocytoid cells quantified. Measurements of monocyte binding were performed on one experimental and one control rat simultaneously, and results presented as the ratio of cells bound by thoracic aortae from the experimental compared with the control rat. With this approach, the ratio of monocyte binding (8% NaCl/chow) was increased in Dahl-S versus Dahl-R rats (1.7 +/- 0.1 v 1.3 +/- 0.1, P < .05), as well as in S-D as compared with F-344 rats (1.7 +/- 0.2 v 1.1 +/-0.1, P < .05). These results provide evidence that hypertension in Dahl-S and fructose-fed S-D rats was associated with changes in the endothelium that favor atherogenesis.

Abstract

This study was initiated to compare the adherence to cultured endothelial cells of mononuclear cells isolated from normotensive and hypertensive individuals.Mononuclear cell binding to endothelium was greater in patients with hypertension (32+/-1 versus 25+/-2; P<0.001) than in normal volunteers. There was a significant relationship (r=0.42, P<0. 01) between mononuclear cell binding and mean arterial pressure, independent of differences in age, sex, and body mass index. A significant relationship also existed between insulin resistance (estimated by the steady-state plasma glucose concentration during the insulin suppression test) and mononuclear cell binding in both the normotensive (r=0.86, P<0.001) and hypertensive (r=0.74, P<0. 001) groups. Furthermore, multiple regression analysis demonstrated an independent relationship (P<0.001) between mononuclear cell binding and both steady-state plasma glucose and hypertensive status.These results indicate that both hypertension and insulin resistance lead to changes in mononuclear cells that increase their adherence to cultured endothelial cells.

Abstract

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA.ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity.These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.

Abstract

We have recently found that administration of L-arginine to hypercholesterolemic rabbits induces regression of preexisting lesions. Others have previously shown that activation of the L-arginine/nitric oxide (NO) synthase pathway can induce apoptosis of vascular cells in vitro. Accordingly, the current study was designed to determine if dietary supplementation of L-arginine induces apoptosis of intimal lesions and if this effect is mediated through the NO synthase pathway.Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water, and the cholesterol diet was continued for 2 weeks, at which time the aortas were harvested for histological studies. L-Arginine treatment increased the number of apoptotic cells (largely macrophages) in the intimal lesions by 3-fold (11.9+/-3.9 vs 3.9+/-1. 4 apoptotic cells/mm2, P<0.01). In subsequent studies, aortas were harvested for ex vivo studies. Aortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of the NO synthase pathway. The tissues were then collected for histological studies and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside (10(-5) mol/L) to the medium caused a time-dependent increase in apoptosis of vascular cells (largely macrophages) in the intimal lesion. L-Arginine (10(-3) mol/L) had an identical effect on apoptosis, which was associated with an increase in nitrogen oxides released into the medium. These effects were not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor L-nitro-arginine (10(-4) mol/L). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-BR cGMP.These results indicate that supplemental L-arginine induces apoptosis of macrophages in intimal lesions by its metabolism to NO, which acts through a cGMP-independent pathway. These studies are consistent with our previous observation that supplementation of dietary arginine induces regression of atheroma in this animal model. These studies provide a rationale for further investigation of the therapeutic potential of manipulating the NO synthase pathway in atherosclerosis.

Abstract

Epidemiological studies have established that diabetes mellitus and hypertension are independent risk factors for atherosclerosis. One of the earliest abnormalities seen in atherogenesis is enhanced monocyte adherence to the endothelium. The mechanisms by which diabetes mellitus or hypertension enhances monocyte-endothelial cell interactions are incompletely characterized. It is not known whether there are additive interactions between these risk factors on endothelial adhesiveness for monocytes. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were fed a normal or fructose-enriched diet. In some cases, animals were injected with streptozotocin (35 mg/kg body weight) to induce diabetes. After 2 weeks, plasma was drawn for biochemical measurements, and thoracic aortas were harvested, opened longitudinally, and exposed to fluorescently labeled mouse monocytoid cells (WEHI 78/24, 2 x 10(6)/mL) for 30 minutes on a rocking platform. Adherent cells were counted by epifluorescence microscopy. WEHI 78/24 binding to aortic segments from SHR animals was elevated compared with segments from WKYs. Fructose feeding alone had no effect on endothelial adhesiveness. When WKYs were made hyperglycemic by STZ injection, monocyte binding was 160% of the control value. Elevated monocyte binding was also observed in aortas derived from SHR animals injected with STZ, indicating an additive effect of hypertension and hyperglycemia. To determine whether alterations in oxidative state played a role in the endothelial adhesiveness, aortic segments were exposed to lucigenin (250 micromol/L) for measurement of superoxide anion. Aortic segments from SHR elaborated 120% more superoxide anion than did controls. Elevated free-radical production was also observed in aortas from diabetic WKYs. Furthermore, thoracic aortas derived from diabetic SHR animals elaborated more superoxide anion than did any of the other groups (374%, P<0.05). Immunohistochemical staining for monocyte chemotactic protein-1 demonstrated increased expression in aortas isolated from diabetic WKY and SHR compared with control vessels. These studies demonstrate that both diabetes and hypertension lead to increased monocyte adherence to the endothelium. This abnormality is associated with increased vascular superoxide production and monocyte chemotactic protein-1 expression. Furthermore, there appears to be an additive interaction between hyperglycemia and hypertension in their effects on endothelial adhesiveness and its determinants.

Abstract

Occlusive vascular disease begins with an alteration of the endothelium, which is characterized by a decrease in nitric oxide (NO) activity. Endogenous NO inhibits many key processes in atherogenesis, including monocyte adherence, platelet activation, and smooth muscle proliferation. The mechanism by which NO activity is reduced in hypercholesterolemia and in other metabolic disorders associated with atherogenesis appears to be multifactorial. It includes increased production of oxygen-derived free radicals, alterations in NO synthase, and the accumulation of endogenous inhibitors (ADMA) of NO synthase. Plasma concentrations of ADMA are elevated in hypercholesterolemic humans. Elevated ADMA concentrations are associated with impaired endothelium-dependent, NO-mediated vasodilatation and reduced urinary nitrate exertion. These effects of ADMA are counteracted by administration of the NO precursor L-arginine. It is likely that basic insights regarding the mechanisms of endothelial dysfunction will lead to new therapeutic strategies for atherosclerosis.

Abstract

Hypercholesterolemia reduces vascular nitric oxide (NO) activity. This dysfunction may promote endothelial monocyte interaction, as NO is a potent inhibitor of cell adhesion. We have previously shown that in hypercholesterolemic (HC) rabbits, chronic oral supplementation of L-arginine (Arg) restores NO activity and inhibits monocyte-endothelial cell interaction, in association with a reduction in atherogenesis. We hypothesized that enhancement of endothelial NO activity in HC humans would reduce monocyte adhesiveness. We used a functional binding assay to assess the adhesiveness of human mononuclear cells (MNCs) ex vivo to determine the effects of hypercholesterolemia and L-arginine administration. MNCs from HC subjects adhered in greater numbers (50% more cells per high-power field; P < .0001) than cells derived from normocholesterolemic (NC) subjects. To determine whether enhancement of endogenous NO activity could inhibit mononuclear cell adhesiveness, in a double-blinded placebo-controlled study, oral arginine HCl (8.4 g/d) was administered to HC subjects. Over a course of 2 weeks, this treatment abolished the increased adhesiveness of HC MNCs (160 +/- 11% versus 104 +/- 5%; before and after 2 weeks of Arg treatment; results expressed as a percentage of the binding values obtained using cells derived from paired NC individuals). By contrast, MNC adhesion remained significantly elevated in placebo-treated HC subjects. To examine whether endothelium-derived NO could act as a paracrine modulator of monocyte behavior, monocytes were exposed to NO donors or cocultered in the presence of endothelial cells exposed to antagonists of NO synthase in the presence or absence of L-arginine. NO donors inhibited monocyte adhesiveness. Furthermore, the adhesiveness of monocytes cocultured with endothelial cells was increased by antagonists of NO synthase; this effect was reversed by L-arginine. This study shows that the adhesiveness of human MNCs is increased by hypercholesterolemia. The increase in adhesiveness was reversed in vivo by administration of the NO precursor L-arginine. NO donors or endothelium-derived NO inhibits the adhesiveness of monocytes in vitro, supporting the hypothesis that the effects of L-arginine are mediated by NO.

Abstract

Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti-L-selectin and anti-alpha4 and beta2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

Abstract

To compare the binding to cultured endothelial cells of mononuclear cells isolated from healthy volunteers and patients with NIDDM.Mononuclear cells were isolated from healthy volunteers (n = 11) and patients with NIDDM (n = 14) and incubated with ECV 304 cells, a human umbilical endothelial cell-derived transformed cell line. Following a period of incubation, the adherence of mononuclear cells to endothelial cells was determined.Adherence of mononuclear cells from patients with NIDDM was significantly greater (P < 0.05) than that of cells isolated from the healthy volunteers, and this difference persisted when adjusted for age, sex, and degree of obesity. Mononuclear cell binding to ECV 304 cells correlated significantly with fasting plasma glucose (r = 0.52, P < 0.01), insulin (r = 0.51, P < 0.01), triglyceride (r = 0.54, P < 0.01), and VLDL (r = 0.54, P < 0.01) and HDL cholesterol (r = -0.45, P < 0.05) levels, but not with either total or LDL cholesterol levels or blood pressure.Since the adherence of mononuclear cells to the endothelium represents the earliest step in atherogenesis, the observation that mononuclear cells from patients with NIDDM bind more avidly to cultured endothelial cells may help explain why accelerated atherosclerosis occurs in patients with NIDDM. The metabolic abnormality, or abnormalities, present in patients with NIDDM that is responsible for the enhanced adhesiveness of mononuclear cells requires further examination.

Abstract

Monocyte chemotactic protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. We and others have demonstrated that NO inhibits monocyte-endothelial cell interactions and atherogenesis. We hypothesize that the antiatherogenic effect of NO may be due in part to its inhibition of MCP-1 expression.Smooth muscle cells (SMCs) were isolated from normal rabbit aortas by the explant method. Cells were then exposed to LPS (10 microg/mL), native LDL, or oxidized LDL (30 microg/mL) for 6 hours. The expression of MCP-1 in SMCs and chemotactic activity in the conditioned medium were induced by lipopolysaccharide (LPS) or by oxidized LDL but not native LDL. The induction of MCP-1 by cytokines or oxidized lipoproteins was associated with an increased generation of superoxide anion by the SMCs and increased activity of the transcriptional protein nuclear factor-kappaB (NFkappaB). The induced expression of MCP-1 and activation of NFkappaB were reduced by previous exposure of the SMCs to the NO donor DETA-NONOate (100 micromol/L) (P

Abstract

Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.

Abstract

We have recently shown that ex vivo gene therapy of rabbit autologous vein grafts with antisense oligodeoxynucleotides (AS ODN) blocking cell cycle regulatory gene expression inhibits not only neointimal hyperplasia, but also diet-induced, accelerated graft atherosclerosis. We observed that these grafts remained free of macrophage invasion and foam cell deposition. Since endothelial dysfunction plays an important role in vascular disease, the current study examined the effect of this genetic engineering strategy on graft endothelial function and its potential relationship to the engineered vessels' resistance to atherosclerosis. Rabbit vein grafts transfected with AS ODN against proliferating cell nuclear antigen (PCNA) and cell division cycle 2 (cdc2) kinase elaborated significantly more nitric oxide and exhibited greater vasorelaxation to both calcium ionophore and acetylcholine than did untreated or control ODN-treated grafts. This preservation of endothelial function was associated with a reduction in superoxide radical generation, vascular cell adhesion molecule-1 (VCAM-1) expression, and monocyte binding activity in grafts in both normal and hypercholesterolemic rabbits. Our data demonstrate that AS ODN arrest of vascular cell cycle progression results in the preservation of normal endothelial phenotype and function, thereby influencing the biology of the vessel wall towards a reduction of its susceptibility to occlusive disease.

Abstract

The present study was designed to test the hypothesis that long-term dietary supplementation with the nitric oxide precursor L-arginine would enhance vascular or platelet-derived nitric oxide activity, or both, and thereby inhibit platelet reactivity in hypercholesterolemic humans.We have shown that reduced vascular activity of nitric oxide in hypercholesterolemic rabbits can be restored by L-arginine supplementation. The improvement in nitric oxide activity is associated with an inhibition of platelet aggregation ex vivo. This effect is most likely due to increased elaboration of endothelium- or platelet-derived nitric oxide, or both, because the inhibition of platelet reactivity was associated with elevation of intraplatelet cyclic guanosine monophosphate and was reversed by the nitric oxide synthase antagonist N-methyl-arginine.In a double-blinded, randomized, placebo-controlled trial, hypercholesterolemic patients were assigned to L-arginine hydrochloride, 8.4 g/day orally, or placebo for 2 weeks. Platelet-rich plasma was obtained for aggregometry induced by collagen (1 to 10 micrograms/ml) at four points: baseline, after 2 weeks of treatment, after a 2-week washout and after a long-term washout of 16 weeks on average. Aggregation was quantified by light transmittance and expressed as a percent transmittance observed with platelet-poor plasma.Compared with normocholesterolemic control subjects, platelets from hypercholesterolemic subjects stimulated with 5 micrograms/ml of collagen showed increased aggregability (68.6% in hypercholesterolemic patients vs. 54.5% in normocholesterolemic control subjects, p < or = 0.02). After 2 weeks of treatment with L-arginine (but not placebo), platelet reactivity was modestly reduced; this effect persisted for 2 weeks after discontinuation of arginine (52.6% in arginine-treated patients vs. 65.1% in normocholesterolemic control subjects, p = 0.07). After 18 weeks (i.e., 16 weeks after discontinuing arginine treatment), the platelets of hypercholesterolemic patients once again became hyperaggregable, and the extent of platelet aggregation was significantly increased compared with the 4-week point (73.6% after vs. 52.6% during arginine treatment, p < 0.01). No significant change in platelet reactivity was seen in placebo-treated hypercholesterolemic patients throughout the study. L-Arginine treatment was well tolerated without side effects.This double-blinded, placebo-controlled study demonstrates that dietary supplementation with L-arginine can modestly attenuate the increased platelet reactivity seen in hypercholesterolemic patients. The data are consistent with our previous studies in hypercholesterolemic animals, demonstrating that L-arginine restores endogenous nitric oxide activity and inhibits platelet aggregation. Enhancement of endogenous nitric oxide activity is a potential novel therapeutic strategy worthy of further study.

Abstract

This study sought to determine whether the alterations in vascular function and structure after balloon injury in hypercholesterolemic rabbits could be inhibited by dietary arginine.Administration of arginine (the nitric oxide [NO] precursor) restores vascular NO activity in hypercholesterolemic animals. We and other investigators have shown that enhancement of vascular NO activity can inhibit myointimal hyperplasia after vascular injury in normocholesterolemic animals.Twenty-eight New Zealand White rabbits received either normal rabbit chow, 0.5% cholesterol diet or 0.5% cholesterol diet plus L-arginine hydrochloride (2.25% wt/vol) in the drinking water. After 6 weeks of dietary intervention, the left iliac artery of each animal was subjected to a balloon injury. Four weeks later, the iliac arteries were harvested for vascular reactivity studies and immunohistochemical analysis.Vascular injury induced intimal thickening that was largely composed of vascular smooth muscle cells and extracellular matrix. In the setting of hypercholesterolemia, vascular injury induced an exuberant myointimal lesion that was augmented by the accumulation of lipid-laden macrophages. Dietary arginine reduced intimal thickening in the injured vessels of hypercholes-terolemic animals and substantially inhibited the accumulation of macrophages in the lesion (from 28% to 5% of the lesion area, p < 0.001).We report that lesions induced by vascular injury in hypercholesterolemic animals are markedly reduced by oral administration of arginine. Moreover, we find that the nature of the lesion is altered, with a striking reduction in the percentage of macrophages comprising the lesion.

Abstract

In the arterial tree, regions exposed to reduced shear stress (low and/or disturbed flow) are predisposed to atherogenesis. Fluid flow is a potent stimulus for the release of endothelium-derived nitric oxide (NO). Because NO inhibits monocyte-endothelial cell interaction, we speculated that the effects of flow in inhibiting atherogenesis might be mediated in part by NO.Confluent monolayers of human aortic endothelial cells were exposed to static or fluid flow conditions for 4 hours. The medium was replaced, and cells were then incubated with native LDL (50 micrograms/mL), oxidized LDL (30 micrograms/mL), or lipopolysaccharide (LPS) (10 ng/mL)+tumor necrosis factor-alpha (TNF-alpha) (10 U/mL) for an additional 4 hours. Functional binding assays using THP-1 monocytes were then performed. Superoxide production by human aortic endothelial cells was monitored by lucigenin chemiluminescence, and expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were quantified by flow cytometry. Whereas native LDL had little effect, incubation with either oxidized LDL or LPS/TNF-alpha significantly increased superoxide production, nuclear factor-kappa B activity, VCAM-1 expression, and endothelial adhesiveness for monocytes. Previous exposure to fluid flow inhibited these sequelae of exposure to cytokines or oxidized lipoprotein. The effect of fluid flow appears to be due in part to shear-induced release of NO, because coincubation with nitro-L-arginine completely abolished these effects of flow. Furthermore, the NO donor PAPA-NONO-ate and 8-Br-cGMP (but not 8-Br-cAMP) mimicked the effects of flow.Previous exposure to fluid flow decreased cytokine- or lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 expression, and monocyte binding; the effects of flow appear to be due to NO. Flow-mediated NO-dependent regulation of oxidant-responsive transcription may influence the site of a lesion.

Abstract

Hypertension is a known clinical risk factor for atherosclerosis. In experimental atherosclerosis, monocyte adhesion to the endothelial surface is enhanced and is considered to be an important early stage in plaque formation. We tested the hypothesis that hypertension enhances monocyte adhesion in experimental atherosclerosis.Twenty-two New Zealand White rabbits were fed an atherogenic diet for 3 weeks to induce plaque formation. Aortic coarctation was created in eight rabbits by wrapping a Dacron band around the midportion of the descending thoracic aorta (stenosis group), whereas six rabbits underwent banding without aortic constriction (no stenosis group). Eight rabbits served as nonoperated controls. Monocyte binding to the aortic endothelial surface was counted with epifluorescent microscopy on standard aortic segments proximal and distal to the band. Immunohistochemistry was performed for the following antibodies: VCAM-1, RAM11, CD11b, and factor VIII.Mean blood pressure was 89 +/- 3 mm Hg in the aorta proximal to the stenosis, compared with 64 +/- 4 mm Hg in the no stenosis group and 74 +/- 3 mm Hg in the control group (p < 0.01). The mean aortic blood pressure gradient across the stenosis was 16 +/- 2 mm Hg in the stenosis group, whereas the aortic blood pressure gradient was 0.2 +/- 0.6 mm Hg in the no stenosis group and -0.3 +/- 0.4 mm Hg in the control group (p < 0.001). Monocyte adhesion to the aortic endothelial surface proximal to the stenosis was increased twofold compared with adhesion to the aorta distal to the stenosis and compared with the proximal aorta in the control group (p < 0.02). The proximal-to-distal aortic ratio of monocyte binding was enhanced in the stenosis group (2.2) compared with the no stenosis (0.76) and control (0.83) groups (p < 0.01). The intima area of the aorta proximal to the stenosis was significantly increased compared with the proximal aortas in the no stenosis and control groups (p < 0.01). RAM11, CD11b, and endothelial VCAM-1 expression were enhanced in the hypertensive region proximal to the stenosis.In the hypertensive region in the aorta proximal to the stenosis, monocyte adhesion and endothelial VCAM-1 expression were increased, with intimal thickening and accumulation of macrophages. These findings suggest that hypertension may promote atherosclerotic plaque formation by enhancing monocyte adhesion.

Abstract

There is increasing evidence that alterations in nitric oxide synthesis are of pathophysiological importance in heart failure. A number of studies have shown altered nitric oxide production by the endothelial constitutive isoform of nitric oxide synthase (NOS), but there is very little information on the role of the inducible isoform.We analyzed inducible NOS (iNOS) expression in ventricular myocardium taken from 11 control subjects (who had died suddenly from noncardiac causes), from 10 donor hearts before implantation, and from 51 patients with heart failure (24 with dilated cardiomyopathy [DCM], 17 with ischemic heart disease [IHD], and 10 with valvular heart disease [VHD]). Reverse transcription-polymerase chain reaction was used to confirm the presence of intact mRNA and to detect expression of iNOS and atrial natriuretic peptide (ANP). ANP was used as a molecular phenotypic marker of ventricular failure. iNOS was expressed in 36 of 51 biopsies (71%) from patients with heart failure and in none of the control patients (P

Abstract

The mechanisms underlying cardiac contractile dysfunction after transplantation remain poorly defined. Previous work has revealed that inducible nitric oxide synthase (iNOS) is expressed in the rat heterotopic cardiac allograft during rejection; resultant overproduction of nitric oxide (NO) might cause cardiac contractile dysfunction via the negative inotropic and cytotoxic actions of NO. In this investigation, we tested the hypothesis that induction of iNOS may occur and be associated with cardiac allograft contractile dysfunction in humans.We prospectively studied 16 patients in the first year after cardiac transplantation at the time of serial surveillance endomyocardial biopsy. Clinical data, the results of biopsy histology, and echocardiographic and Doppler evaluation of left ventricular systolic and diastolic function were recorded. Total RNA was extracted from biopsy specimens, and mRNA for beta-actin, detected by reverse transcription-polymerase chain reaction (RT-PCR) using human specific primers, was used as a constitutive gene control; iNOS mRNA was similarly detected by RT-PCR using human specific primers. iNOS protein was detected in biopsy frozen sections by immunofluorescence. Myocardial cGMP was measured by radioimmunoassay, and serum nitrogen oxide levels (NOx = NO2 + NO3) were measured by chemiluminescence. iNOS mRNA was detected in allograft myocardium at some point in each patient and in 59 of 123 biopsies (48%) overall. In individual patients, iNOS mRNA expression was episodic and time dependent; the frequency of expression was highest during the first 180 days after transplant (P = .0006). iNOS protein associated with iNOS mRNA was detected by immunofluorescence in cardiac myocytes. iNOS mRNA expression was not related to the ISHLT histological grade of rejection or to serum levels of NOx but was associated with increased levels of myocardial cGMP (P = .01) and with both systolic (P = .024) and diastolic (P = .006) left ventricular contractile dysfunction measured by echocardiography and Doppler.These data support a relation between iNOS mRNA expression and contractile dysfunction in the human cardiac allograft.

Abstract

We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine inhibits atherogenesis in the hypercholesterolemic rabbit. However, the effect of supplemental arginine on preexisting lesions is not known and was the focus of the present study. New Zealand White rabbits received normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently, L-arginine (2.25% in drinking water; ARG group) or vehicle (CHOL group) was administered for an additional 13 weeks, while the high-cholesterol diet was continued. Thoracic aortae were harvested at weeks 10, 14, 18, or 23. Rings of aorta were used to assess NO-dependent vasodilation to acetylcholine. Maximal relaxation to acetylcholine in the CHOL rabbits became progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). Planimetry of the luminal surface of the aortae from CHOL animals revealed a progressive increase in lesion surface area from 30.3% (at week 10) to 56.5% (by week 23). By contrast, animals in the ARG groups manifested improved endothelium-dependent relaxation associated with a reduction of lesion surface area at 14 and 18 weeks. The arginine-induced improvement in endothelium-dependent relaxation was associated with an increased generation of vascular NO and a reduced generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had persistent improvement in NO-dependent vasodilation and exhibited a further reduction of lesion surface area tc 5.4%. We conclude that hypercholesterolemia induces a progressive loss of NO-dependent vasodilation associated with progressive intimal lesion formation. Administration of L-arginine to animals with preexisting intimal lesions augments vascular NO elaboration, reduces superoxide anion generation, and is associated with a reduction in lesion surface area. This is the first demonstration that restoration of NO activity can induce regression of preexisting intimal lesions and provides evidence that L-arginine therapy may be of potential clinical benefit.

Abstract

Shear stress increases the release of nitric oxide (NO) by endothelial cells (ECs). We and others have provided evidence that endothelium-derived NO inhibits monocyte adhesion to the vessel wall. We therefore hypothesized that previous exposure to shear stress would inhibit endothelial adhesiveness for monocytes by virtue of its effect to increase NO release.Confluent monolayers of bovine aortic endothelial cells, human aortic endothelial cells, or human venous endothelial cells were exposed to laminar fluid flow. Culture media were collected for measurement of NO (by chemiluminescence) and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha. NOx and 6-keto-prostaglandin F1 alpha accumulated in the conditioned medium during laminar fluid flow from 30 minutes to 24 hours in a time-dependent fashion. In another set of studies, ECs previously exposed to flow or to static conditions were washed with Hanks' buffer and exposed to THP-1 cells for 30 minutes. Adherent cells were counted by microscopy. Previous exposure to flow reduced endothelial adhesiveness for monocytes by 50% (P < .05). The effect of flow on endothelial adhesiveness occurred within 30 minutes. This effect was abrogated by nitro-L-arginine (an antagonist of NO synthesis), as well as by tetraethylammonium ion (an antagonist of the flow-activated potassium channel); the effects of these inhibitors were reversed by the NO donor SPM-5185. Although the cyclo-oxygenase inhibitor indomethacin totally inhibited the flow-induced production of prostacyclin by ECs, it minimally affected adherence of THP-1 cells. The early effect of flow on endothelial adhesiveness was not mediated by alterations in the expression of the endothelial adhesion molecules VCAM-1 or ICAM-1 as assessed by fluorescent activated cell sorting.Shear stress alters endothelial adhesiveness for monocytes; at early time points, this effect is largely due to flow-stimulated release of NO and, to a lesser extent, prostacyclin. This effect of flow occurs within 30 minutes and is probably due to alterations in the signal transduction or activation state (rather than the expression) of endothelial adhesion molecules.

Abstract

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

Abstract

We investigated the effect of dietary supplementation of L-arginine (L-Arg), the precursor of endothelial nitric oxide (NO), on endothelium-dependent and endothelium-independent vascular responses, as well as vascular structure, in the abdominal aorta of hypercholesterolemic rabbits. Rabbits were fed (a) normal rabbit chow, (b) 1% cholesterol diet, or (c) 1% cholesterol diet supplemented with 2.25% L-Arg HCl in drinking water. After 10 weeks, the abdominal aorta was harvested for study of vascular reactivity and histomorphometry. L-Arg did not affect serum cholesterol levels. Histomorphometric analysis demonstrated an eightfold reduction in intimal thickening in the abdominal aorta of the arginine-supplemented hypercholesterolemic rabbits. By contrast, the effects on vascular reactivity were subtle. Contraction to norepinephrine (NE) was not altered by hypercholesterolemia or L-Arg. Contraction to acetylcholine (ACh) was increased in hypercholesterolemic animals; this was normalized by dietary arginine supplementation. Relaxation to nitroglycerin (NTG) was not altered by hypercholesterolemia but was attenuated in the arginine-supplemented rabbits. Endothelium-dependent relaxation to ACh was impaired in both hypercholesterolemic groups. Dietary L-Arg has a dramatic antiatherogenic effect in hypercholesterolemic rabbits. This effect is associated with rather slight changes in vascular reactivity that are suggestive of a slight increase in NO elaboration by the endothelium. The discordance between the effects of dietary arginine on vascular structure and reactivity suggests that the antiatherogenic effects of the NO precursor may not be mediated entirely by its effect on the endothelium.

Abstract

Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.

Abstract

We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine normalizes NO-dependent vasodilation and markedly inhibits atherogenesis in a hypercholesterolemic rabbit model. We hypothesized that this antiatherogenic effect is due to modulation of endothelial adhesiveness by endothelium-derived NO.New Zealand White rabbits were fed normal chow (Cont), a high-cholesterol diet (Chol), a high-cholesterol diet supplemented with L-arginine (Arg), or a normal diet supplemented with the NO synthase antagonist L-nitroarginine (L-NA) for 2 weeks. In additional studies, some animals receiving L-NA were also treated with hydralazine to normalize blood pressure. After 2 weeks, thoracic aortas were harvested, opened longitudinally, and placed in a culture dish with the endothelial surface exposed to medium containing WEHI 78/24 cells, a monocytoid cell line. After incubation with the monocytoid cells for 30 minutes on a rocking platform, the aortic segments were washed repeatedly to remove nonadherent cells and adherent cells counted by epifluorescent microscopy. Monocytoid cell binding to aortic endothelium was significantly increased in Chol (P < .001 versus Cont); binding was markedly reduced in arginine-fed hypercholesterolemic animals (P < .05, Arg versus Chol). Monocytoid cell binding to aortic endothelium was also significantly increased in L-NA (P < .05); hydralazine normalized blood pressure but did not reduce monocytoid cell binding. To confirm that alterations in NO activity modulate endothelial cell-monocyte interaction, the release of nitrogen oxides (NOx) by thoracic aortas was assessed by a chemiluminescent technique. The concentration of NOx in the conditioned medium from segments of Arg thoracic aortas was significantly greater than that from Cont aortas, whereas that from L-NA aortas was significantly less.Hypercholesterolemia enhances the adhesiveness of aortic endothelium for monocytes; this effect is attenuated by dietary L-arginine. Conversely, inhibition of NO synthesis enhances monocyte binding. The results suggest that endothelium-derived NO plays an important role in regulating the endothelial adhesiveness for monocytes. Alterations in NO activity may play a critical role in atherogenesis.

Abstract

This study was designed to test the hypothesis that long-term oral supplementation of dietary L-arginine (to provide a sustained elevation of nitric oxide activity) would inhibit atherogenesis in hypercholesterolemic rabbits, as assessed by histomorphometric measurements.Endothelium-derived nitric oxide inhibits a number of processes that are critical in atherogenesis. Hypercholesterolemia reduces endothelial nitric oxide activity, and we postulate that this may promote atherogenesis. This reduction in nitric oxide activity can be reversed acutely by intravenous infusion of L-arginine, the precursor of nitric oxide. We show that dietary supplementation of L-arginine abrogates the development of coronary atheroma in hypercholesterolemic rabbits.Male New Zealand White rabbits were fed normal rabbit chow, 1% cholesterol chow or 1% cholesterol chow with dietary arginine or methionine supplementation to increase their intake of these amino acids sixfold. After 1 or 10 weeks of dietary intervention, the left main and left anterior descending coronary arteries were harvested for histologic study. Plasma cholesterol measurements were elevated to the same degree in all groups of rabbits receiving the 1% cholesterol diet, whereas plasma arginine levels were doubled in the arginine-treated group. High density lipoprotein (HDL) cholesterol values were not affected by arginine treatment.In rabbits receiving the 1% cholesterol diet, with or without methionine supplementation, light and electron microscopy revealed a marked increase from 1 to 10 weeks in the intimal accumulation of macrophages, associated with an increase in the intimal area of the left main coronary artery. By contrast, in arginine-treated hypercholesterolemic rabbits, there was a near absence of adherent monocytes and tissue macrophages and no progression of intimal thickness from 1 to 10 weeks.Dietary supplements of L-arginine prevent intimal thickening in the coronary arteries of hypercholesterolemic rabbits. This antiatherogenic effect is not due to an alteration in plasma total cholesterol, HDL cholesterol or caloric or nitrogen balance. The data are consistent with the hypothesis that nitric oxide has antiatherogenic properties.

Abstract

The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.